Predictive biomarkers for response to coenzyme q10 treatment in pancreatic cancer

Abstract

The disclosure describes the use of predictive markers in methods for prognosing the response to a Coenzyme Q10 treatment for pancreatic cancer in a subject, and methods for method of treating pancreatic cancer based on the prognostic information.

Description

PREDICTIVE BIOMARKERS FOR RESPONSE TO COENZYME Q10 TREATMENTIN PANCREATIC CANCERRELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 623,119, filed on January 19, 2024, the entire contents of which are incorporated hereby by reference.BACKGROUND OF THE INVENTION

[0002] Pancreatic ductal adenocarcinoma (PDAC) or pancreatic cancer, is a highly aggressive disease with mortality rate nearly equal to incidence. As about 80% of patients are initially diagnosed with advanced disease, prognosis of pancreatic cancer is extremely poor. According to the American Cancer Society, in 2015 pancreatic cancer was ranked as the fourth leading cause of cancer-related mortality in the United States; however, it is projected to become the second leading cause of cancer death by 2030. See Cancer Res. 74: 2913-21 (2014).

[0003] Although recent research has vastly increased our understanding of many of the molecular mechanisms of tumorigenesis and has provided numerous new avenues for the treatment of cancer, standard treatments for most malignancies remain gross resection, chemotherapy, and radiotherapy. While increasingly successful, each of these treatments still causes numerous undesired side effects. For example, surgery results in pain, traumatic injury to healthy tissue, and scarring. Radiotherapy and chemotherapy cause nausea, immune suppression, gastric ulceration and secondary tumorigenesis. There is a need to identify markers that can indicate outcome of a treatment regimen in order for both clinicians and patients to decide on the best treatment option for patients.SUMMARY OF THE INVENTION

[0004] The present invention is based, at least in part, on the discovery that certain markers (e.g., proteins, lipids and metabolites) are differentially regulated in pancreatic cancer subjects that responded to a drug treatment, e.g., Coenzyme Q10. In particular, the invention is based on the surprising discovery that a higher level of SERPINA5,Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and PA-P18:O / 18:O in samples of pancreatic cancer patients indicates that the patients are responsive to a drug treatment, e.g., Coenzyme Q10, and a lower level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in samples of pancreatic cancer patients indicates that the patients are responsive to a drug treatment, e.g., Coenzyme Q10.

[0005] Accordingly, in one aspect, the present invention provides a method for prognosing a response to Coenzyme Q10 treatment for pancreatic cancer in a subject, comprising: (a) detecting the level of one or more markers in a biological sample from the subject, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l; and (b) comparing the level of the one or more markers in the biological sample with a predetermined threshold value; wherein an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and / or PA- P18:O / 18:O relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment; and / or wherein a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

[0006] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0007] In some embodiments, the Coenzyme Q10 treatment is administered by intravenous administration. In some embodiments, the Coenzyme Q10 treatment is administered by continuous infusion.

[0008] In some embodiments, an increase in the expression level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PL18:0 / 20:2, and / or PA- P18:O / 18:O relative to the predetermined threshold value, and / or a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will exhibit stable disease in response to the Coenzyme Q10 treatment.

[0009] In some embodiments, a second drug is administered. In some embodiments, the second drug is gemcitabine.

[0010] In some embodiments, the response to the Coenzyme Q10 treatment comprises no change or a decrease in tumor size. In some embodiments, the response to the Coenzyme Q10 treatment comprises an increase in overall days of survival.

[0011] In some embodiments, the biological sample comprises a blood sample or a component thereof. In some embodiments, the sample comprises a buffy coat sample. In some embodiments, the sample comprises a plasma sample.

[0012] In some embodiments, the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers is determined.

[0013] In some embodiments, the level of the one or more markers is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

[0014] In some embodiments, the level of the one or more markers is detected by determining the level of its corresponding mRNA in the biological sample. In some embodiments, the level of the one or more markers is detected by determining the level of its protein in the biological sample.

[0015] In some embodiments, the method further comprises selecting and / or administering a treatment regimen based on the prediction of the Coenzyme Q10 treatment in the subject.

[0016] In some embodiments, the treatment regimen comprises further monitoring the subject for progression of pancreatic cancer.

[0017] In some embodiments, the treatment regimen is selected from the group consisting of (a) radiation therapy, (b) chemotherapy, (c) surgery, (d) hormone therapy, (e) antibody therapy, (f) immunotherapy, (g) cytokine therapy, (h) growth factor therapy, (i) watchful waiting, and (i) any combination of (a)-(i).

[0018] In some embodiments, the treatment regimen comprises administering Coenzyme Q10, optionally in combination with gemcitabine.

[0019] In some embodiments, the subject has been previously diagnosed with pancreatic cancer.

[0020] In another aspect, the present invention provides a method for prognosing a response to Coenzyme Q10 treatment in a subject, wherein the prognosis is determined concurrently with the diagnosis of pancreatic cancer in the subject, comprising: (a) diagnosing the subject with pancreatic cancer; (b) detecting the level of one or more markers in a biological sample from the subject, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1; and (c) comparing the level of the one or more markers in the biological sample with a predetermined threshold value; wherein an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, and / or PA-P18:O / 18:O relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment; and / or wherein a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

[0021] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0022] In some embodiments, the Coenzyme Q10 treatment is administered by intravenous administration. In some embodiments, the Coenzyme Q10 treatment is administered by continuous infusion.

[0023] In some embodiments, a second drug is administered. In some embodiments, the second drug is gemcitabine.

[0024] In some embodiments, the response to the Coenzyme Q10 treatment comprises no change or a decrease in tumor size. In some embodiments, the response to the Coenzyme Q10 treatment comprises an increase in overall days of survival.

[0025] In some embodiments, the biological sample comprises a blood sample or a component thereof. In some embodiments, the sample comprises a buffy coat sample. In some embodiments, the sample comprises a plasma sample.

[0026] In some embodiments, the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers, is determined.

[0027] In some embodiments, the level of the one or more markers is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

[0028] In some embodiments, the level of the one or more markers is detected by determining the level of its corresponding mRNA in the biological sample. In some embodiments, the level of the one or more markers is detected by determining the level of its protein in the biological sample.

[0029] In some embodiments, the method further comprises selecting and / or administering a treatment regimen based on the prediction of the Coenzyme Q10 treatment in the subject.

[0030] In some embodiments, the treatment regimen comprises further monitoring of the subject for progression of cancer. In some embodiments, the treatment regimen is selected from the group consisting of (a) radiation therapy, (b) chemotherapy, (c) surgery, (d) hormone therapy, (e) antibody therapy, (f) immunotherapy, (g) cytokine therapy, (h) growth factor therapy, (i) watchful waiting, and (i) any combination of (a)-(i).

[0031] In some embodiments, the treatment regimen comprises administering Coenzyme Q10, optionally in combination with gemcitabine.

[0032] In one aspect, the present invention provides a method for identifying an agent that modulates pancreatic cancer progression, comprising; (a) contacting a pancreatic cancer cell with a test compound, (b) determining the level of a marker in the cell, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1; (c) identifying an agent that modulates the level of the marker in the cell, thereby identifying an agent that modulates pancreatic cancer progression.

[0033] In some embodiments, the test compound is a small molecule, an antibody, or a nucleic acid inhibitor.

[0034] In another aspect, the present invention provides a compound identified by the method as described herein.

[0035] In one aspect, the present invention provides a method of treating pancreatic cancer in a subject, comprising administering to the subject a modulator of a marker,wherein the marker is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1.

[0036] In some embodiments, the modulator increases the marker level or activity. In some embodiments, the modulator decreases the marker level or activity.

[0037] In one aspect the present invention provides a kit for detecting a marker in a biological sample from a subject having pancreatic cancer, comprising one or more reagents for measuring the level of the marker in the biological sample from the subject, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1, and a set of instructions for measuring the level of the marker.

[0038] In some embodiments, the reagent is an antibody that binds to the marker or an oligonucleotide that is complementary to the corresponding mRNA of the marker.

[0039] In some embodiments, the instructions set forth an immunoassay, ELISA, or mass spectrometry assay for detecting the level of the marker in the biological sample. In some embodiments, the instructions set forth an amplification reaction for assaying the level of the mRNA in the biological sample corresponding to the marker. In some embodiments, the instructions set forth a hybridization assay for detecting the level of the mRNA in the biological sample corresponding to the marker. In some embodiments, the instructions further set forth comparing the level of the marker in the biological sample from the subject to a predetermined threshold value of the marker.

[0040] In some embodiments, the marker comprises one or more markers with an increased level when compared to a predetermined threshold value, and / or one or more markers with a decreased level when compared to a predetermined threshold value.

[0041] In one aspect, the present invention provides a panel for use in a method of prognosing a response to a Coenzyme Q10 treatment for pancreatic cancer in a subject, the panel comprising one or more detection reagents, wherein each detection reagent is specific for the detection of a marker, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PL18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1.

[0042] In another aspect, the present invention provides a kit comprising the panel as described herein and a set of instructions for obtaining prognosis information based on a level of the marker.

[0043] In one aspect, the present invention provides a method for detecting a marker useful for prognosing a response to Coenzyme Q10 treatment for pancreatic cancer, comprising: (a) obtaining a sample from a subject diagnosed with pancreatic cancer; and (b) detecting the level of the marker in the biological sample, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1 .

[0044] In some embodiments, the biological sample comprises a blood sample or a component thereof. In some embodiments, the sample comprises a buffy coat sample. In some embodiments, the sample comprises a plasma sample.

[0045] In some embodiments, the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers, is determined.

[0046] In some embodiments, the level of the marker is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

[0047] In some embodiments, the level of the marker is detected by determining the level of its corresponding mRNA in the biological sample. In some embodiments, the level of the one or more markers is detected by determining the level of its protein in the biological sample.

[0048] In another aspect, the present invention provides a method of treating pancreatic cancer in a subject comprising: (a) obtaining a biological sample from the subject; (b) submitting the biological sample to obtain prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2; and (c) administering a therapeutically effective amount ofCoenzyme Q10 to the subject if the level of the one or more markers is above or below a predetermined threshold value.

[0049] In one aspect, the present invention provides a method of treating pancreatic cancer in a subject comprising: (a) obtaining prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2; and (b) administering a therapeutically effective amount of Coenzyme Q10 to the subject if the level of the one or more markers is above or below a predetermined threshold value.

[0050] In another aspect, the present invention provides a method of treating pancreatic cancer in a subject suspected of having pancreatic cancer, the method comprising: (a) obtaining a biological sample from the subject for use in identifying prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2; (b) measuring the level of the one or more markers in the biological sample; and (c) recommending to a healthcare provider to administer Coenzyme Q10 if the level of the one or more markers in the biological sample is above or below a predetermined threshold value.

[0051] In some embodiments, the subject is responsive to the Coenzyme Q10 treatment if the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, and / or PA-P18:O / 18:O is above the predetermined threshold value.

[0052] In some embodiments, the subject is responsive to the Coenzyme Q10 treatment if the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 is below the predetermined threshold value.

[0053] In some embodiments, the methods further comprise administering a therapeutically effective amount of gemcitabine.

[0054] In some embodiments, the biological sample comprises a blood sample or a component thereof. In some embodiments, the sample comprises a buffy coat sample. In some embodiments, the sample comprises a plasma sample.

[0055] In some embodiments, the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers, is determined.

[0056] In some embodiments, the level of the one or more markers is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

[0057] In some embodiments, the level of the one or more markers is detected by determining the level of its corresponding mRNA in the biological sample. In some embodiments, the level of the one or more markers is detected by determining the level of its protein in the biological sample.BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 shows the differential expression of protein, lipid, and metabolomic markers measured in the ATC population at baseline (prior to treatment with Coenzyme Q10). Differential expression analysis considered patient response to be either (A) “Best response during study,” which refers to the best RECIST status determined during the entire Coenzyme Q10 treatment, or (B) “Patient status at Cycle 2”, which refers to the RECIST status determined at Cycle 2 of Coenzyme Q10 treatment. For each molecular marker, the most significant clinical response is shown. Hence, SERPINA5, 2-keto- isovalerate, Proteosome 20S subunit alpha 4, SL-7-HDHA, PI- 18:0 / 20:2, and PC- 38:8 / 0-38:1 are shown in terms of differential expression of “Best Response During Study,” while Vitronectin, Lumican, PL18:0 / 22:4, and PA-P18:O / 18:O are shown in terms of differential expression of “Patient Status at Cycle 2.”DETAILED DESCRIPTION OF THE INVENTIONA. OVERVIEW

[0059] Some cancer has very low survival rate, such as pancreatic ductal adenocarcinoma (PDAC) with a 5 year survival rate less than 8%. Markers that can indicate outcome of a treatment regimen will better inform clinicians on which treatment options to choose for their patients, as well as inform patients on whether to choose no treatment for a better life quality or commit to a therapy with detrimental side effects to the overall health. The present invention addresses this need for markers by providing the use of biomarkers, i.e.one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, for the identification of subjects having a good response and overall survival outcome to a drug treatment, e.g. Coenzyme Q10.

[0060] As presently described herein, the present invention is based, at least in part, on the surprising discovery that the one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l are differentially regulated in pancreatic cancer subjects that responded to a drug treatment, e.g., Coenzyme Q10. In particular, the invention is based on the surprising discovery that a higher level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PL 18:0 / 22:4, PI- 18:0 / 20:2, and / or PA-P18:O / 18:O in samples of pancreatic cancer patients indicates that the patients are responsive to a drug treatment, e.g., Coenzyme Q10, and a lower level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38:l in samples of pancreatic cancer patients indicates that the patients are responsive to a drug treatment, e.g., Coenzyme Q10.

[0061] Accordingly, the invention provides methods for prognosing and / or monitoring (e.g., monitoring of disease progression or treatment) outcome of a drug treatment to a cancer in a subject, e.g., outcome of PDAC after treatment with Coenzyme-QlO.

[0062] In one embodiment, these one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof, can serve as useful prognostic biomarkers, serving to inform on the likely development or progression of pancreatic cancer, e.g. PDAC, in a subject. In still another embodiment, these one or more markers, e.g. SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof, can serve as useful predictive biomarkers for helping to assess the likely response of pancreatic cancer, e.g. PDAC, to a particular treatment, e.g., Coenzyme Q10, optionally in combination with gemcitabine.

[0063] The following is a detailed description of the invention provided to aid those skilled in the art in practicing the present invention. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0064] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials arc now described. All publications mentioned herein arc incorporated herein by reference to disclose and described the methods and / or materials in connection with which the publications are cited.B. DEFINITIONS

[0065] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references, the entire disclosures of which are incorporated herein by reference, provide one of skill with a general definition of many of the terms (unless defined otherwise herein) used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, the Harper Collins Dictionary of Biology (1991). Generally, the procedures of molecular biology methods described or inherent herein and the like are common methods used in the ail. Such standard techniques can be found in reference manuals such as for example Sambrook et al. , (2000, Molecular Cloning— A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al., (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New-York).

[0066] The following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0067] As used herein, the singular forms "a", "and", and "the" include plural references unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning.

[0068] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0069] As used herein, the term “amplification" refers to any known in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. Known in vitro amplification methods include, e.g., transcription- mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand-displacement amplification (SDA including multiple strand-displacement amplification method (MSDA)). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Q-P-replicase (e.g., Kramer et al., U.S. Patent No. 4,786,600). PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat.App. Pub. No. 0320 308). SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Patent. No. 5,422,252). Two other known strand-displacement amplification methods do not require endonuclease nicking (Dattagupta et al., U.S. Patent. No. 6,087,133 and U.S. Patent. No. 6,124,120 (MSDA)). Those skilled in the art will understand that the oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase, (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl.Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000, Molecular Cloning— A Laboratory Manual, Third Edition, CSH Laboratories). As commonly known in the ait, the oligos are designed to bind to a complementary sequence under selected conditions.

[0070] As used herein, the term “antigen” refers to a molecule, e.g., a peptide, polypeptide, protein, fragment, or other biological moiety, which elicits an antibody response in a subject, or is recognized and bound by an antibody.

[0071] As used herein, the term "marker" is a biological molecule, or a panel of biological molecules, for example, SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PL18:0 / 22:4, PL18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7- HDHA, and PC-38:8 / O-38:l, or any combination thereof, whose altered level in a tissue, cell or body fluid as compared to its level in tissue, cell or body fluid from, e.g., a subject with a disease that progressed to a more advanced stage after receiving a treatment, is associated with having good response to a treatment (e.g., decrease in tumor size, no increase in tumor size, increased overall time of survival, increased time to progression). Examples of biomarkers include, for example, polypeptides, peptides, polypeptide fragments, proteins, antibodies, hormones, polynucleotides, RNA or RNA fragments, microRNA (miRNAs), lipids, metabolites, or polysaccharides. In an embodiment, the marker is detected in a body fluid, e.g., blood or plasma. In a preferred embodiment, the marker is detected in the buffy coat of blood. In a preferred embodiment, the marker isdetected in plasma. In certain embodiments, the blood, plasma or buffy coat sample can be further processed to remove abundant proteins or proteins that are not marker proteins prior to analysis.

[0072] The term “marker” as used herein, also includes any one or more pathological or clinical feature or parameter. For example, as described herein, a marker includes clinical parameters such as, e.g., cancer stage, e.g., stage 0, stage I, stage II, stage III, stage IV, tumor size, age, performance status, or any clinical and / or patient-related health data, for example, data obtained from an Electronic Medical Record (e.g., collection of electronic health information about individual patients or populations relating to various types of data, such as, demographics, medical history, laboratory test results, radiology images, vital signs, personal statistics like weight, and billing infoimation).

[0073] Preferably, a marker of the present invention is modulated (e.g., increased or decreased level) in a biological sample from a subject or a group of subjects having a first phenotype (e.g., having cancer progression) as compared to a biological sample from a subject or group of subjects having a second phenotype (e.g., not having cancer progression, e.g., a control). A biomarker may be differentially present at any level, but is generally present at a level that is increased relative to normal or control levels by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more; or is generally present at a level that is decreased relative to normal or control levels by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent). A biomarker is preferably differentially present at a level that is statistically significant (e.g., a p-value less than 0.05 and / or a q-value of less than 0.10 as determined using either Welch's T-test or Wilcoxon's rank-sum Test). As such, the difference between the level of a biomarker ofthe present invention and a corresponding control or reference value can be a statistically significant positive or negative value.

[0074] The term "pancreatic cancer" or “pancreatic ductal adenocarcinoma (PDAC)” as used interchangeably herein, refers to any type of cancerous or precancerous tissues arising from normal tissues of the pancreas, including, but not limited to, exocrine pancreatic cancer, PanIN lesions, pancreatic ductal adenocarcinoma or pancreatic adenocarcinoma. Other types of pancreatic tumors include acinar-cell carcinoma, serous cystadenoma and pancreatic endocrine tumors.

[0075] As used herein, the term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallcl to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0076] The term “control sample” or “control,” as used herein, refers to any clinically relevant comparative sample, including, for example, a sample from a healthy subject not afflicted with an oncological disorder, e.g., pancreatic cancer, or a sample from a subject from an earlier time point, e.g., prior to treatment, an earlier tumor assessment time point, at an earlier stage of treatment. In some embodiments, a control sample is a sampleobtained from a subject having pancreatic cancer prior to treatment of the subject with Coenzyme Q10, and who did not ultimately have a good response to Coenzyme Q10 treatment. A control sample can be a purified sample, protein, and / or nucleic acid provided with a kit. Such control samples can be diluted, for example, in a dilution series to allow for quantitative measurement of levels of analytes, e.g., markers, in test samples. A control sample may include a sample derived from one or more subjects. A control sample may also be a sample made at an earlier time point from the subject to be assessed. For example, the control sample could be a sample taken from the subject to be assessed before the onset of an oncological disorder, e.g., pancreatic cancer, at an earlier stage of disease, or before the administration of treatment or of a portion of treatment. The control sample may also be a sample from an animal model, or from a tissue or cell line derived from the animal model of oncological disorder, e.g., pancreatic cancer. The level of activity or expression of one or more markers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 markers) in a control sample consists of a group of measurements that may be determined, e.g., based on any appropriate statistical measurement, such as, for example, measures of central tendency including average, median, or modal values. In one embodiment, “different from a control” is preferably statistically significantly different from a control.

[0077] As used herein, “changed, altered, increased or decreased as compared to a control” sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator (e.g., marker) to be detected at a level that is statistically different, e.g., increased or decreased, as compared to a control sample (e.g., from a normal, untreated, or abnormal state control sample). Changed as compared to control can also include a difference in the rate of change of the level of one or more markers obtained in a series of at least two subject samples obtained over time. Determination of statistical significance is within the ability of those skilled in the art and can include any acceptable means for determining and / or measuring statistical significance, such as, for example, the number of standard deviations from the mean that constitute a positive or negative result, an increase in the detected level of a biomarker in a sample (e.g. , pancreatic cancer sample) versus a control or healthy sample, wherein the increase is above some threshold value, or a decrease in the detected level of a biomarker in a sample (e.g., pancreaticcancer sample) versus a control or healthy sample, wherein the decrease is below some threshold value.

[0078] The term “control level” refers to an accepted or pre-determined level of a marker in a subject sample. A control level can be a range of values. Marker levels can be compared to a single control value, to a range of control values, to the upper level of normal, or to the lower level of normal as appropriate for the assay.

[0079] In one embodiment, the control is a standardized control, such as, for example, a control which is predetermined using an average of the levels of expression of one or more markers from a population of biologically relevant control subjects, e.g. subject whose cancer did not respond to a drug treatment, e.g., Coenzyme Q10, and continued to progress. In certain embodiments, the control can be from a subject, or a population of subjects, having an abnormal pancreatic state, e.g. acute pancreatitis, chronic pancreatitis, hereditary pancreatitis. It is understood that not all markers will have different levels for each of the abnormal pancreatic states listed. It is understood that a combination of marker levels may be most useful to distinguish between cancer subjects, e.g. PDAC subjects, that will likely respond to a drug (e.g., Coenzyme Q10) treatment (e.g. decreased tumor size, prolonged survival time) from cancer subjects that will not benefit from a drug treatment (e.g., Coenzyme Q10). Further, marker levels in biological samples can be compared to more than one control sample (e.g., normal, abnormal, from the same subject, from a population control). Marker levels can be used in combination with other signs or symptoms of an abnormal state to provide a prognosis for the subject.

[0080] A control can also be a sample from a subject at an earlier time point, e.g., a baseline level prior to suspected progression of disease, before the diagnosis of a disease, at an earlier assessment time point during watchful waiting, before the treatment with a specific agent (e.g., chemotherapy, hormone therapy) or intervention (e.g., radiation, surgery). In certain embodiments, a change in the level of the marker in a subject can be more significant than the absolute level of a marker, e.g., as compared to control.

[0081] As used herein, “detecting”, “detection”, “determining”, and the like are understood to refer to an assay performed for identification of one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-PI 8:0 / 18:0, Proteosome 20S subunit alpha 4,SL-7-HDHA, and PC-38:8 / O-38:l. The amount of marker expression or activity detected in the sample can be none or below the level of detection of the assay or method.

[0082] As used herein, the term "DNA" or "RNA" molecule or sequence (as well as sometimes the term "oligonucleotide") refers to a molecule comprised generally of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and / or cytosine (C). In "RNA", T is replaced by uracil (U).

[0083] The terms “disorders”, “diseases”, and “abnormal state” are used inclusively and refer to any deviation from the normal structure or function of any pail, organ, or system of the body (or any combination thereof). A specific disease is manifested by characteristic symptoms and signs, including biological, chemical, and physical changes, and is often associated with a variety of other factors including, but not limited to, demographic, environmental, employment, genetic, and medically historical factors. An early stage disease state includes a state wherein one or more physical symptoms arc not yet detectable. Certain characteristic signs, symptoms, and related factors can be quantitated through a variety of methods to yield important diagnostic or prognostic information. As used herein the disorder, disease, or abnormal state is an abnormal pancreatic state, including pancreatitis, pancreatic neoplasm, or a non-cancerous pancreatic mass,.

[0084] As used herein, a sample obtained at an “earlier time point” is a sample that was obtained at a sufficient time in the past such that clinically relevant information could be obtained in the sample from the earlier time point as compared to the later time point. In certain embodiments, an earlier time point is at least four weeks earlier. In certain embodiments, an earlier time point is at least six weeks earlier. In certain embodiments, an earlier time point is at least two months earlier, hi certain embodiments, an earlier time point is at least three months earlier. In certain embodiments, an earlier time point is at least six months earlier. In certain embodiments, an earlier time point is at least nine months earlier. In certain embodiments, an earlier time point is at least one year’ earlier. Multiple subject samples (e.g., 3, 4, 5, 6, 7, or more) can be obtained at regular or irregular intervals over time and analyzed for trends in changes in marker levels.Appropriate intervals for testing for a particular subject can be determined by one of skill in the art based on ordinary considerations.

[0085] The term “expression” is used herein to mean the process by which a polypeptide is produced from DNA. The process involves the transcription of the gene into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which used, “expression” may refer to the production of RNA, or protein, or both.

[0086] As used herein, “fold change ratio” or “FC ratio” refers to a change, e.g., increase or decrease, of the expression or level of a marker, e.g., one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l. In some embodiments, the FC ratio is greater than 1, which indicates an up-regulation or increase in the expression or level of the marker. In other embodiments, the FC ratio is less than 1, indicating a down-regulation or decrease in the expression or level of the marker. FC ratio can also be calculated and expressed as a Log unit. When the FC ratio is expressed as a Log FC value, a Log FC value greater than 0 is equivalent to an FC ratio greater than 1, indicating an up-regulation or increase in the expression or level of the marker. Alternatively, a Log FC value less than 0 is equivalent to an FC ratio less than 1, indicating a down-regulation or decrease in the expression or level of the marker.

[0087] As used herein, “greater predictive value” is understood as an assay that has significantly greater sensitivity and / or specificity, preferably greater sensitivity and specificity, than the test to which it is compared. The predictive value of a test can be determined using an ROC analysis. In an ROC analysis, a test that provides perfect discrimination or accuracy between normal and disease states would have an area under the curve (AUC)=1, whereas a very poor test that provides no better discrimination than random chance would have AUC=0.5. As used herein, a test with a greater predictive value will have a statistically improved AUC as compared to another assay. The assays are performed in an appropriate subject population.

[0088] A “higher level of expression”, “higher level”, “increased level,” and the like of a marker refers to a level in a test sample that is greater than the standard error of the assay employed to assess the level, and is preferably at least 25% more, at least 50% more, at least 75% more, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times the level of the marker in acontrol sample (e.g., sample from a healthy subject not having the marker associated disease) and preferably, the average level of the marker or markers in several control samples.

[0089] As used herein, the term “hybridization,” as in "nucleic acid hybridization," refers generally to the hybridization of two single- stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double- stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook el al., 2000, supra and Ausubel et al., 1994, supra, or further in Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington D.C., (1985)) and are commonly known in the ail. In the case of a hybridization to a nitrocellulose filter (or other such support like nylon), as for example in the well-known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at a temperature representative of the desired stringency condition (60-65°C for high stringency, 50-60°C for moderate stringency and 40-45°C for low stringency conditions) with a labeled probe in a solution containing high salt (6xSSC or 5xSSPE), 5xDenhardt's solution, 0.5% SDS, and 100 pg / ml denatured carrier DNA (e.g., salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2xSSC / 0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency). The salt and SDS concentration of the washing solutions may also be adjusted to accommodate for the desired stringency. The selected temperature and salt concentration is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well-known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al., 2000, supra). Other protocols or commercially available hybridization kits (e.g., ExpressHyb® from BD Biosciences Clonetech) using different annealing and washing solutions can also be used as well known in the ait. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditionsmay be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and allelic variants of these molecules.Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).

[0090] As used herein, the term "identical" or "percent identity" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 65% identity, preferably, 70-95% identity, more preferably at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 60% to 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 amino acids or nucleotides in length, more preferably, over a region that is about 50 to 100 amino acids or nucleotides in length. Those having skill in the art will know how to determine percent identity between / among sequences using, for example, algorithms such as those based on CLUSTALW computerprogram (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art. Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul Nucl. Acids Res. 25 (1977), 3389-3402). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11 , an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci., USA, 89, (1989), 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. Moreover, the present invention also relates to nucleic acid molecules the sequence of which is degenerate in comparison with the sequence of an above-described hybridizing molecule. When used in accordance with the present invention the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid. The present invention also relates to nucleic acid molecules which comprise one or more mutations or deletions, and to nucleic acid molecules which hybridize to one of the herein described nucleic acid molecules, which show (a) mutation(s) or (a) deletion(s).

[0091] The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to.”

[0092] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

[0093] As used herein, a "label" refers to a molecular moiety or compound that can be detected or can lead to a detectable signal. A label is joined, directly or indirectly, to a molecule, such as an antibody, a nucleic acid probe or the protein / antigen or nucleic acidto be detected (e.g., an amplified sequence). Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker" or bridging moiety, such as oligonucleotide(s) or small molecule carbon chains, which is either directly or indirectly labeled. Bridging moieties may amplify a detectable signal. Labels can include any detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound). Preferably, the label on a labeled probe is detectable in a homogeneous assay system, i.e., in a mixture, the bound label exhibits a detectable change compared to an unbound label.

[0094] The terms “level of expression of a gene”, “gene expression level”, “level of a marker”, and the like refer to the level of mRNA, as well as prc-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s) and degradation products, or the level of protein, encoded by the gene in the cell, or the level of lipid or metabolite. The “level” of one of more biomarkers means the absolute or relative amount or concentration of the biomarker in the sample.

[0095] A “lower level of expression” or “lower level” or “decreased level” of a marker refers to a level in a test sample that is less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the level of the marker in a control sample (e.g., sample from a healthy subjects not having the marker associated disease) and preferably, the average level of the marker in several control samples.

[0096] The term “modulation” refers to upregulation (i.e., activation or stimulation), down-regulation (i.e., inhibition or suppression) of a response (e.g., level of a marker), or the two in combination or apart. A “modulator” is a compound or molecule that modulates, and may be, e.g., an agonist, antagonist, activator, stimulator, suppressor, or inhibitor.

[0097] As used herein, "nucleic acid molecule" or "polynucleotides", refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA) and chimeras thereof. The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]). Conventional ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) are included in the term "nucleic acid" and polynucleotides as are analogs thereof. A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as "peptide nucleic acids" (PNA); Hydig-Hielsen et al., PCT Inti Pub. No. WO 95 / 32305), phosphoro thioate linkages, methylphosphonate linkages or combinations thereof. Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2' methoxy substitutions (containing a 2'-O-methylribofuranosyl moiety; see PCT No. WO 98 / 02582) and / or 2' halide substitutions. Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 1 1th ed., 1992), or known derivatives of purine or pyrimidine bases (sec, Cook, PCT Int'l Pub. No. WO 93 / 13121) or "abasic" residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs). An "isolated nucleic acid molecule", as is generally understood and used herein, refers to a polymer of nucleotides, and includes, but should not limited to DNA and RNA. The "isolated" nucleic acid molecule is purified from its natural in vivo state, obtained by cloning or chemically synthesized.

[0098] As used herein, the term “obtaining” is understood herein as manufacturing, purchasing, or otherwise coming into possession of.

[0099] As used herein, "oligonucleotides" or "oligos" define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthesized chemically or derived by cloning according to well-known methods. While they are usually in a single-stranded form, they can be in a double- stranded form and even contain a "regulatory region". They can contain natural rare or synthetic nucleotides. They can bedesigned to enhance a chosen criteria like stability for example. Chimeras of deoxyribonucleotides and ribonucleotides may also be within the scope of the present invention.

[0100] As used herein, “one or more” or “at least one of’ is understood as each value 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and any value greater than 10.

[0101] The term “or” is used inclusively herein to mean, and is used interchangeably with, the term “and / or,” unless context clearly indicates otherwise.

[0102] As used herein, “patient” or “subject” can mean either a human or non-human animal, preferably a mammal. By “subject” is meant any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds. A human subject may be referred to as a patient. It should be noted that clinical observations described herein were made with human subjects and, in at least some embodiments, the subjects arc human.

[0103] As used herein, “preventing” or “prevention” refers to a reduction in risk of acquiring a disease or disorder (z.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Prevention does not require that the disease or condition never occurs in the subject. Prevention includes delaying the onset or severity of the disease or condition.

[0104] As used herein, a “predetermined threshold value” or “threshold value” of a biomarker refers to the level of the biomarker (e.g., the expression level or quantity (e.g., ng / ml) in a biological sample) in a corresponding control sample or group of control samples obtained from, for example, a normal and healthy subject or population of subjects not afflicted with pancreatic cancer, a subject or population of subjects afflicted with pancreatic cancer, e.g. PDAC, a sample from a subject or population of subjects whose cancer has progressed without receiving any treatment e.g., Coenzyme Q10), a subject whose cancer has progressed after treatment (e.g., Coenzyme Q10), or a subject from an earlier time point, e.g., prior to treatment, an earlier tumor assessment time point, at an earlier stage of cancer, or prior to onset of cancer. In one embodiment, a predetermined threshold value is a level of the biomarker in a sample (or samples) obtained from a subject (or population of subjects) afflicted with pancreatic cancer, e.g.,PDAC, before treatment of the subject(s) with Coenzyme Q10, and who ultimately did not have a good response to Coenzyme Q10 treatment. The predetermined threshold value may be determined prior to or concurrently with measurement of marker levels in a biological sample. The control sample may be from the same subject at a previous time or from different subjects.

[0105] As used herein, a "probe" is meant to include a nucleic acid oligomer or oligonucleotide that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence). A probe's "target" generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or "base pairing." Sequences that are "sufficiently complementary" allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary. A probe may be labeled or unlabeled. A probe can be produced by molecular cloning of a specific DNA sequence or it can also be synthesized. Numerous primers and probes which can be designed and used in the context of the present invention can be readily determined by a person of ordinary skill in the ail to which the present invention pertains.

[0106] As used herein, the terminology "prognosis", "staging" and "determination of aggressiveness" are defined herein as the prediction of the degree of severity of the pancreatic cancer and of its evolution as well as the prospect of recovery as anticipated from usual course of the disease. According to the present invention, once the aggressiveness of the pancreatic cancer has been determined appropriate methods of treatments can be chosen. As used herein, “prognosing a response to Coenzyme Q10 treatment” refers to the prediction of response of a pancreatic cancer in a subject to treatment with Coenzyme Q10 (e.g., treatment with Coenzyme Q10 alone or in combination with one or more additional anti-cancer therapeutic agents).

[0107] As used herein, “prophylactic” or “therapeutic” treatment refers to administration to the subject of one or more agents or interventions to provide the desired clinical effect. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing at least one sign or symptom of the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic i.e., it is intended to diminish, ameliorate, or maintain at least one sign or symptom of the existing unwanted condition or side effects therefrom).

[0108] As used herein, a "reference level" of a biomarker means a level of the bio marker that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof. A "reference level" of a biomarkcr may be an absolute or relative amount or concentration of the biomarkcr, a presence or absence of the biomarker, a range of amount or concentration of the biomarker, a minimum and / or maximum amount or concentration of the biomarker, a mean amount or concentration of the biomarker, and / or a median amount or concentration of the biomarker; and, in addition, "reference levels" of combinations of biomarkers may also be ratios of absolute or relative amounts or concentrations of two or more biomarkers with respect to each other. Appropriate positive and negative reference levels of biomarkers for a particular disease state, phenotype, or lack thereof may be determined by measuring levels of desired biomarkers in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between biomarker levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of biomarkers in biological samples (e.g., LC-MS, GC-MS, etc.), where the levels of biomarkers may differ based on the specific technique that is used.

[0109] As used herein, “sample” or “biological sample” includes a specimen or culture obtained from any source. Biological samples can be obtained from blood (including any blood product, such as whole blood, plasma, serum, buffy coat, or specific types of cellsT1of the blood), urine, saliva, seminal fluid, and the like. Biological samples also include tissue samples, such as biopsy tissues or pathological tissues that have previously been fixed (e.g., formaline snap frozen, cytological processing, etc.).

[0110] As use herein, the phrase "specific binding" or "specifically binding" when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure ( / .<?., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A," the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0111] The phrase “specific identification” is understood as detection of a marker of interest with sufficiently low background of the assay and cross-reactivity of the reagents used such that the detection method is diagnostically useful. In certain embodiments, reagents for specific identification of a marker bind to only one isoform of the marker. In certain embodiments, reagents for specific identification of a marker bind to more than one isoform of the marker. In certain embodiments, reagents for specific identification of a marker bind to all known isoforms of the marker.

[0112] As used herein, the phrase "subject suspected of having cancer" refers to a subject that presents one or more symptoms indicative of a cancer or is being screened for a cancer (e.g., during a routine physical). A subject suspected of having cancer may also have one or more risk factors. A subject suspected of having cancer has generally not been tested for cancer. However, a "subject suspected of having cancer" encompasses an individual who has received an initial diagnosis but for whom the stage of cancer is not known. The term further includes people who once had cancer (e.g., an individual in remission).

[0113] The term “such as” is used herein to mean, and is used interchangeably, with the phrase “such as but not limited to.”

[0114] The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., cancer). Test compoundscomprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. In some embodiments of the present invention, test compounds include antisense compounds.

[0115] The term “therapeutic effect” refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or in the enhancement of desirable physical or mental development and conditions in an animal or human. A therapeutic effect can be understood as a decrease in tumor growth, decrease in tumor growth rate, stabilization or decrease in tumor burden, stabilization or reduction in tumor size, stabilization or decrease in tumor malignancy, increase in tumor apoptosis, and / or a decrease in tumor angiogenesis.

[0116] As used herein, “therapeutically effective amount” means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease, e.g., the amount of such a substance that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment, e.g., is sufficient to ameliorate at least one sign or symptom of the disease, e.g., to prevent progression of the disease or condition, e.g., prevent tumor growth, decrease tumor size, induce tumor cell apoptosis, reduce tumor angiogenesis, prevent metastasis. When administered for preventing a disease, the amount is sufficient to avoid or delay onset of the disease. The “therapeutically effective amount” will vary depending on the compound, its therapeutic index, solubility, the disease and its severity and the age, weight, etc., of the patient to be treated, and the like. For example, certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment. Administration of a therapeutically effective amount of a compound may require the administration of more than one dose of the compound.

[0117] A "transcribed polynucleotide" or “nucleotide transcript” is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or having a high percentage of identity (e.g., at least 80% identity)with all or a portion of a mature mRNA made by transcription of a marker of the invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.

[0118] As used herein, “treatment,” particularly “active treatment,” refers to performing an intervention to treat pancreatic cancer in a subject. Depending on the stage and type of cancer, treatment options include, but are not limited to, therapy to, e.g., reduce at least one of the growth rate or tumor burden, reduce or maintain the tumor size or the malignancy e.g., likelihood of metastasis) of the tumor, increase apoptosis in the tumor by one or more of administration of a therapeutic agent, e.g., chemotherapy, hormone therapy, stimulate the immune system to eliminate cancer cells, e.g., immunotherapy; administration of radiation therapy (e.g., pellet implantation, brachytherapy), or surgical resection of the tumor, or any combination thereof appropriate for treatment of the subject based on grade and stage of the tumor and other routine considerations. Active treatment is distinguished from “watchful waiting” (i.e., not active treatment) in which the subject is monitored, but no interventions are performed. Watchful waiting can include administration of agents that alter effects caused by the recurrence that are not administered to alter the growth or pathology of the recurrence itself.

[0119] The recitation of a listing of chemical group(s) in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0120] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0121] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0122] Reference will now be made in detail to exemplary embodiments of the invention. While the invention will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the invention to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.C. BIOMARKERS OF THE INVENTION

[0123] The invention at hand is based, at least in part, on the surprising discovery that the one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P 18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l are differentially regulated between cohorts of pancreatic cancer patients that positively responded to treatment with Coenzyme Q10 and those that did not. hi particular’, the invention is based on the surprising discovery that certain markers are either elevated (e.g. SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and / or PA- P18:0 / 18 :0) or depressed (e.g. Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC- 38:8 / 0-38:1) in the plasma or buffy coat of pancreatic cancer patients, particularly PDAC patients, that responded well to treatment with Coenzyme Q10 in comparison to patients that did not respond to the treatment.

[0124] Accordingly, the invention provides methods for prognosing and / or monitoring (e.g., monitoring of disease progression or treatment) pancreatic cancer development or lack thereof in a pancreatic cancer subject after a drug treatment, e.g., Coenzyme Q10.

[0125] The invention also provides methods for treating or for adjusting treatment regimens based on prognostic information relating to the levels of one or more of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PL18:0 / 22:4, PL18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof, of a subject having pancreatic cancer, e.g., PDAC. The invention further provides panels and kits for practicing the methods of the invention.

[0126] The present invention provides new markers and combinations of markers for use in predicting outcome of treatment with Coenzyme-QlO in a cancer subject. Thesemarkers are particularly useful in screening for PDAC subjects that will likely respond positively to Coenzyme Q10. Therefore, the present invention provides methods for selecting a subject having pancreatic cancer for treatment with Coenzyme Q10 (e.g., Coenzyme Q10 alone or in combination with another anti-cancer therapeutic).

[0127] The markers of the invention include, but are not limited to one or more markers among SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof.

[0128] In one embodiment, these one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof, can serve as useful prognostic biomarkers, serving to inform on the likely development or progression of pancreatic cancer, e.g. PDAC, in a subject. In still another embodiment, these one or more markers, e.g. SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof, can serve as useful predictive biomarkers for helping to assess the likely response of pancreatic cancer, e.g. PDAC, to a particular treatment, e.g., Coenzyme Q10, optionally in combination with gemcitabine.

[0129] In some embodiments of the present invention, other biomarkers can be used in connection with the methods of the present invention. As used herein, the term “one or more biomarkers” or “at least one of’ is intended to mean that one or more e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof, are assayed, and, in various embodiments, more than one other biomarker and in various combinations may be assayed.

[0130] Methods, kits, and panels provided herein include any combination of e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 markers, selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O,Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, or any combination thereof.

[0131] The markers of the invention are meant to encompass any measurable characteristic that reflects in a quantitative or qualitative manner the physiological state of an organism, e.g., whether the organism’s PDAC is progressing. The physiological state of an organism is inclusive of any disease or non-disease state, e.g., a subject is having PDAC or a subject is healthy. Said another way, the markers of the invention include characteristics that can be objectively measured and evaluated as indicators of normal processes, pathogenic processes, or pharmaco logic responses to a therapeutic intervention, including, in particular, progression of cancer. Examples of markers include, for example, polypeptides, peptides, polypeptide fragments, proteins, antibodies, hormones, polynucleotides, RNA or RNA fragments, microRNA (miRNAs), lipids (e.g. structural lipids or signaling lipids), polysaccharides, and other bodily metabolites that are indicative and / or predictive of the development of an oncological disease.

[0132] In other embodiments, the present invention also involves the analysis and consideration of any clinical and / or patient-related health data, for example, data obtained from an Electronic Medical Record (e.g., collection of electronic health information about individual patients or populations relating to various types of data, such as, demographics, medical history, medication and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics like age and weight, and billing information).

[0133] In certain embodiments, the marker, e.g. marker of responders to Coenzyme Q10 treatment, is SERPINA5 (also known as Serpin Family A Member 5, PROCI, PAI3, Protein C Inhibitor, PLANH3, PCI, Plasminogen Activator Inhibitor III), which is used herein to refer to both the gene and the protein, in both processed and unprocessed forms, unless clearly indicated otherwise by context. The NCBI gene ID for SERPINA5 is 5104 and detailed information can be found at the NCBI website (incorporated herein by reference in the version available on the filing date of the application to which this application claims priority). The human SERPINA5 transcript is listed under accession number NM_000624.6.

[0134] In certain embodiments, an increase in the level of SERPINA5 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

[0135] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is Vitronectin (also known as VTN, VN, Serum Spreading Factor, Complement S-Protein, Somatomedin B, V75, Epibolin), which is used herein to refer to both the gene and the protein, in both processed and unprocessed forms, unless clearly indicated otherwise by context. The NCBI gene ID for Vitronectin is 7448 and detailed information can be found at the NCBI website (incorporated herein by reference in the version available on the filing date of the application to which this application claims priority). The human Vitronectin transcript is listed under accession number NM_000638.4.

[0136] In certain embodiments, an increase in the level of Vitronectin relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0137] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is Lumican (also known as SLRR2D, LDC, Keratan Sulfate Proteoglycan Lumican, Epididymis Secretory Sperm Binding Protein), which is used herein to refer to both the gene and the protein, in both processed and unprocessed forms, unless clearly indicated otherwise by context. The NCBI gene ID for Lumican is 4060 and detailed information can be found at the NCBI website (incorporated herein by reference in the version available on the filing date of the application to which this application claims priority). The human Lumican transcript is listed under accession number NM_002345.4.

[0138] In certain embodiments, an increase in the level of Lumican relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0139] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is Proteosome 20S subunit alpha 4 (also known as PSMA4, HC9, HsT17706, Multicatalytic Endopeptidase Complex Subunit C9, PSC9, Proteasome Component C9), which is used herein to refer to both the gene and the protein, in both processed and unprocessed forms, unless clearly indicated otherwise by context. The NCBI gene ID forPSMA4 is 5685 and detailed information can be found at the NCBI website (incorporated herein by reference in the version available on the filing date of the application to which this application claims priority). The human PSMA4 transcript is listed under accession number NM_002789.6.

[0140] In certain embodiments, a decrease in the level of Proteosome 20S subunit alpha 4 relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0141] Each GenBank number is incorporated herein by reference in the version available on the filing date of the application to which this application claims priority. The protein markers are not limited to the protein sequences set forth in the GenBank Accession Numbers or sequence listing.

[0142] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is a metabolite, for example, 2-kcto-isovalcratc. In certain embodiments, an increase in the level of 2-keto-isovalerate relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0143] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is a lipid molecule, for example, Pl-18:0 / 22:4. In certain embodiments, an increase in the level of PI- 18:0 / 22:4 relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0144] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is a lipid molecule, for example, PI-18:0 / 20:2. In certain embodiments, an increase in the level of PI- 18:0 / 20:2 relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0145] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is a lipid molecule, for example, PA-P18:O / 18:O. In certain embodiments, an increase in the level of PA-P18:O / 18:O relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0146] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is a lipid molecule, for example, SL-7-HDHA. In certain embodiments, a decrease in the level of SL-7-HDHA relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0147] In certain embodiments, the marker, e.g. marker of responders to a drug treatment, is a lipid molecule, for example, PC-38:8 / O-38:l. hi certain embodiments, a decrease in the level of PC-38: 8 / 0-38:1 relative to the predetermined threshold value indicates that the subject will be responsive to the drug treatment.

[0148] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least one of the markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least one marker with a predetermined threshold value, and (3) determining if the level of the at least one marker is above or below a certain threshold level. If the level of the at least one marker is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g. Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least one marker from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0149] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least two of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least two markers with predetermined threshold values, and (3) determining if the level of at least two markers is above or below certain threshold levels. If the level of the at least two markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g., Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least two markers from the group consisting ofSERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0150] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least three of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least three markers with predetermined threshold values, and (3) determining if the level of the at least three markers is above or below certain threshold levels. If the level of the at least three markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g., Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least three markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0151] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least four of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-PI 8:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least four markers with predetermined threshold values, and (3) determining if the level of the at least four markers is above or below certain threshold levels. If the level of the at least four markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g., Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least four markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4,PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38:1.

[0152] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least five of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least five markers with predetermined threshold values, and (3) determining if the level of the at least five markers is above or below certain threshold levels. If the level of the at least five markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g.. Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least five markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38:1.

[0153] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least six of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-PI 8:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least six markers with predetermined threshold values, and (3) determining if the level of the at least six markers is above or below certain threshold levels. If the level of the at least six markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g., Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least six markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4,PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38:1.

[0154] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least seven of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least seven markers with predetermined threshold values, and (3) determining if the level of the at least seven markers is above or below certain threshold levels. If the level of the at least seven markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g., Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least seven markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0155] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least eight of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PL18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least eight markers with predetermined threshold values, and (3) determining if the level of the at least eight markers is above or below certain threshold levels. If the level of the at least eight markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g., Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least eight markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0156] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least nine of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least nine markers with predetermined threshold values, and (3) determining if the level of the at least nine markers is above or below certain threshold levels. If the level of the at least nine markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g.. Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least nine markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38:1.

[0157] In certain embodiments, the prognostic signature is obtained by (1) detecting the level of at least ten of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-PI 8:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l in a biological sample, (2) comparing the level of the at least ten markers with predetermined threshold values, and (3) determining if the level of the at least ten markers is above or below certain threshold levels. If the level of the at least ten markers is above or below the threshold level, then the prognostic signature is predictive or indicative of a pancreatic cancer subject who will be responsive to a drug treatment, e.g., Coenzyme Q10 treatment. In certain embodiments, the prognostic signature can be determined based on an algorithm or computer program that predicts whether the biological sample is from a subject who will be responsive to a drug treatment based on the level of the at least ten markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4,PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38:1.

[0158] Moreover, drag treatment responder profile or signature may be obtained by detecting at least one of the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, in combination with at least one other marker, or more preferably, with at least two other markers, or still more preferably, with at least three other markers, or even more preferably with at least four other markers. Still further, the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PL18:0 / 22:4, PL18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l, in certain embodiments, may be used in combination with at least five other markers, or at least six other markers, or at least seven other markers, or at least eight other markers, or at least nine other markers, or at least ten other markers, or at least eleven other markers, or at least twelve other markers, or at least thirteen other markers, or at least fourteen other markers, or at least fifteen other markers, or at least sixteen other markers, or at least seventeen other markers, or at least eighteen other markers, or at least nineteen other markers, or at least twenty other markers. Further still, the markers from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4, PI- 18:0 / 20:2, PA-P18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38:1 may be used in combination with a multitude of other markers, including, for example, with between about 20-50 other markers, or between 50-100, or between 100-500, or between 500-1000, or between 1000-10,000 or markers or more.

[0159] In certain embodiments, the markers of the invention can include variant sequences. More particularly, certain binding agents / reagents used for detecting certain of the markers of the invention can bind and / or identify variants of these certain markers of the invention. As used herein, the term "variant" encompasses nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identity to a sequence disclosed herein. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.

[0160] Variant sequences generally differ from the specifically identified sequence only by conservative substitutions, deletions or modifications. As used herein, a "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the ait of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1 ) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phc; (4) lys, arg, his; and (5) phc, tyr, trp, his. Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[0161] Polypeptide and polynucleotide sequences may be aligned, and percentages of identical amino acids or nucleotides in a specified region may be determined against another polypeptide or polynucleotide sequence, using computer algorithms that are publicly available. The percentage identity of a polynucleotide or polypeptide sequence is determined by aligning polynucleotide and polypeptide sequences using appropriate algorithms, such as BLASTN or BLASTP, respectively, set to default parameters; identifying the number of identical nucleic or amino acids over the aligned portions; dividing the number of identical nucleic or amino acids by the total number of nucleic oramino acids of the polynucleotide or polypeptide of the present invention; and then multiplying by 100 to determine the percentage identity.

[0162] Two exemplary algorithms for aligning and identifying the identity of polynucleotide sequences are the BLASTN and FASTA algorithms. The alignment and identity of polypeptide sequences may be examined using the BLASTP algorithm. BLASTX and FASTX algorithms compare nucleotide query sequences translated in all reading frames against polypeptide sequences. The FASTA and FASTX algorithms are described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and in Pearson, Methods in Enzymol. 183:63-98, 1990. The FASTA software package is available from the University of Virginia, Charlottesville, Va. 22906-9025. The FASTA algorithm, set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of polynucleotide variants. The readme files for FASTA and FASTX Version 2.0x that arc distributed with the algorithms describe the use of the algorithms and describe the default parameters.

[0163] The BLASTN software is available on the NCBI anonymous FTP server and is available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894. The BLASTN algorithm Version 2.0.6 [Sep. 10, 1998] and Version 2.0.11 [Jan. 20, 2000] set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, is described at NCBI's website and in the publication of Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, 1997.

[0164] In an alternative embodiment, variant polypeptides are encoded by polynucleotide sequences that hybridize to a disclosed polynucleotide under stringent conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM, and preferably less than about 200 mM. Hybridization temperatures can be as low as 5°C, but are generally greater than about 22°C, more preferably greater than about 30°C, and most preferably greater than about 37°C. Longer DNA fragments may require higherhybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. An example of "stringent conditions" is prewashing in a solution of 6XSSC, 0.2% SDS; hybridizing at 65°C, 6XSSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1XSSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2XSSC, 0.1% SDS at 65°C.

[0165] The invention provides for the use of various combinations and sub-combinations of markers. It is understood that any single marker or combination of the markers provided herein can be used in the invention unless clearly indicated otherwise.D. BIOLOGICAL SAMPLES

[0166] The present invention may be practiced with any suitable biological sample that potentially contains, expresses, includes, a detectable disease biomarker, e.g., a lipid biomarker, a polypeptide biomarker, a nucleic acid biomarker, a mRNA biomarker, a microRNA biomarker, or a metabolite biomarker. For example, the biological sample may be obtained from sources that include whole blood, serum, urine, plasma, buffy coat, diseased and / or healthy organ tissue, for example, biopsy of pancreas or pancreatic tumor. Preferably, the biological sample is plasma, serum, buffy coat, or urine.

[0167] The methods of the invention may be applied to the study of any pancreas tissue sample, i.e., a sample of pancreas tissue or fluid, as well as cells (or their progeny) isolated from such tissue or fluid. In another embodiment, the present invention may be practiced with any suitable pancreas tissue samples which are freshly isolated or which have been frozen or stored after having been collected from a subject, or archival tissue samples, for example, with known diagnosis, treatment, and / or outcome history. Pancreas tissue may be collected by any non-invasive means, such as, for example, fine needle aspiration and needle biopsy, or alternatively, by an invasive method, including, for example, surgical biopsy.

[0168] The inventive methods may be performed at the single cell level e.g., isolation and testing of cancerous cells from the pancreas tissue sample). However, the inventive methods may also be performed using a sample comprising many cells, where the assayis "averaging" expression over the entire collection of cells and tissue present in the sample. Preferably, there is enough of the pancreas tissue sample to accurately and reliably determine the expression levels of interest, hr certain embodiments, multiple samples may be taken from the same pancreas tissue in order to obtain a representative sampling of the tissue. In addition, sufficient biological material can be obtained in order to perform duplicate, triplicate or further rounds of testing.

[0169] Any commercial device or system for isolating and / or obtaining pancreas tissue and / or blood or other biological products, and / or for processing said materials prior to conducting a detection reaction is contemplated.

[0170] In certain embodiments, the present invention relates to detecting biomarker nucleic acid molecules (e.g., mRNA encoding the protein markers). In such embodiments, RNA can be extracted from a biological sample, e.g., a pancreas tissue sample, before analysis. Methods of RNA extraction arc well known in the art (sec, for example, J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd Ed., Cold Spring Harbour Laboratory Press: New York). Most methods of RNA isolation from bodily fluids or tissues are based on the disruption of the tissue in the presence of protein denaturants to quickly and effectively inactivate RNases. Generally, RNA isolation reagents comprise, among other components, guanidinium thiocyanate and / or beta-mercaptoethanol, which are known to act as RNase inhibitors. Isolated total RNA is then further purified from the protein contaminants and concentrated by selective ethanol precipitations, phenol / chloroform extractions followed by isopropanol precipitation (see, for example, P. Chomczynski and N. Sacchi, Anal. Biochem., 1987, 162: 156-159) or cesium chloride, lithium chloride or cesium trifluoroacetate gradient centrifugations.

[0171] Numerous different and versatile kits can be used to extract RNA (z.e., total RNA or mRNA) from bodily fluids or tissues (e.g., pancreas tissue samples) and are commercially available from, for example, Ambion, Inc. (Austin, Tex.), Amersham Biosciences (Piscataway, N.J.), BD Biosciences Clontech (Palo Alto, Calif.), BioRad Laboratories (Hercules, Calif.), G1BCO BRL (Gaithersburg, Md.), and Giagen, Inc. (Valencia, Calif.). User Guides that describe in great detail the protocol to be followed are usually included in all these kits. Sensitivity, processing time and cost may bedifferent from one kit to another. One of ordinary skill in the art can easily select the kit(s) most appropriate for a particular situation.

[0172] In certain embodiments, after extraction, mRNA is amplified, and transcribed into cDNA, which can then serve as template for multiple rounds of transcription by the appropriate RNA polymerase. Amplification methods are well known in the ait (see, for example, A. R. Kimmel and S. L. Berger, Methods Enzymol. 1987, 152: 307-316; J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd Ed., Cold Spring Harbour Laboratory Press: New York; "Short Protocols in Molecular Biology", F. M. Ausubel (Ed.), 2002, 5. sup. th Ed., John Wiley & Sons; U.S. Pat. Nos. 4,683,195;4,683,202 and 4,800,159). Reverse transcription reactions may be carried out using nonspecific primers, such as an anchored oligo-dT primer, or random sequence primers, or using a target-specific primer complementary to the RNA for each genetic probe being monitored, or using thermostable DNA polymerases (such as avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase).

[0173] In certain embodiments, the RNA isolated from the sample (e.g., plasma, buffy coat, or pancreas tissue sample) (for example, after amplification and / or conversion to cDNA or cRNA) is labeled with a detectable agent before being analyzed. The role of a detectable agent is to facilitate detection of RNA or to allow visualization of hybridized nucleic acid fragments (e.g., nucleic acid fragments hybridized to genetic probes in an array-based assay). Preferably, the detectable agent is selected such that it generates a signal which can be measured and whose intensity is related to the amount of labeled nucleic acids present in the sample being analyzed. In array-based analysis methods, the detectable agent is also preferably selected such that it generates a localized signal, thereby allowing spatial resolution of the signal from each spot on the array.

[0174] Methods for labeling nucleic acid molecules are well-known in the art. For a review of labeling protocols, label detection techniques and recent developments in the field, see, for example, L. J. Kricka, Ann. Clin. Biochem. 2002, 39: 114-129; R. P. van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol. 1994, 35: 135-153. Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachment of fluorescent dyes (see, for example, L. M. Smith et al., Nucl. Acids Res. 1985, 13: 2399-2412) or of enzymes (see, for example, B. A.Connoly and P. Rider, Nucl. Acids. Res. 1985, 13: 4485-4502); chemical modifications of nucleic acid fragments making them detectable immunochemically or by other affinity reactions (see, for example, T. R. Broker et al., Nucl. Acids Res. 1978, 5: 363-384; E. A. Bayer et al., Methods of Biochem. Analysis, 1980, 26: 1-65; R. Langer et al., Proc. Natl. Acad. Sci. USA, 1981, 78: 6633-6637; R. W. Richardson et al., Nucl. Acids Res. 1983, 11: 6167-6184; D. J. Brigati et al., Virol. 1983, 126: 32-50; P. Tchen et al., Proc. Natl Acad. Sci. USA, 1984, 81: 3466-3470; J. E. Landegent et al., Exp. Cell Res. 1984, 15: 61-62; and A. H. Hopman et al., Exp. Cell Res. 1987, 169: 357-368); and enzyme- mediated labeling methods, such as random priming, nick translation, PCR and tailing with terminal transferase (for a review on enzymatic labeling, see, for example, J. Temsamani and S. Agrawal, Mol. Biotechnol. 1996, 5: 223-232).

[0175] Any of a wide variety of detectable agents can be used in the practice of the present invention. Suitable detectable agents include, but arc not limited to: various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.

[0176] However, in some embodiments, the expression levels are determined by detecting the expression of a gene product (e.g., protein) thereby eliminating the need to obtain a genetic sample e.g., RNA) from the pancreas tissue sample.

[0177] In still other embodiments, the present invention relates to preparing a prediction model for pancreas and / or the likelihood of relapse of pancreatic cancer by preparing a model for pancreatic cancer based on measuring the biomarkers of the invention in known control samples. More particularly, the present invention relates in some embodiments to preparing a predictive model by evaluating the biomarkers of the invention, i.e., the markers selected from the group consisting of SERP1NA5, Vitronectin, Lumican, 2-keto-isovalerate, PL18:0 / 22:4, PL18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0178] The skilled person will appreciate that patient tissue samples containing pancreas cells or pancreatic cancer cells may be used in the methods of the present invention including, but not limited to those aimed at predicting relapse probability. In these embodiments, the level of expression of the signature gene can be assessed by assessing the amount, e.g. absolute amount or concentration, of a signature gene product, e.g., protein and RNA transcript encoded by the signature gene and fragments of the protein and RNA transcript) in a sample, e.g., stool and / or blood obtained from a patient. The sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g. fixation, storage, freezing, lysis, homogenization, DNA or RNA extraction, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of the signature gene product in the sample.

[0179] The invention further relates to the preparation of a model for pancreatic cancer or pancreatic cancer relapse by evaluating the biomarkers of the invention in known samples of pancreatic cancer. More particularly, the present invention relates to a pancreatic cancer model for diagnosing and / or monitoring and / or prognosing pancreatic cancer or pancreatic cancer relapse using the biomarkers of the invention, i.e.. the markers selected from the group consisting of SERP1NA5, Vitronectin, Lumican, 2-keto-isovalerate, Pl- 18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0180] In the methods of the invention aimed at preparing a model for pancreatic cancer or pancreatitis prediction, it is understood that the particular clinical outcome associated with each sample contributing to the model preferably should be known. Consequently, the model can be established using archived tissue samples. In the methods of the invention aimed at preparing a model for pancreatic cancer prediction, total RNA can be generally extracted from the source material of interest, generally an archived tissue such as a formalin-fixed, paraffin-embedded tissue, and subsequently purified. Methods for obtaining robust and reproducible gene expression patterns from archived tissues, including formalin-fixed, paraffin-embedded (FFPE) tissues are taught in U.S.Publ. No. 2004 / 0259105, which is incorporated herein by reference in its entirety. Commercial kits and protocols for RNA extraction from FFPE tissues arc available including, for example, ROCHE High Pure RNA Paraffin Kit (Roche) MasterPureTM Complete DNA and RNAPurification Kit (EPICENTREOMadison, Wis.); Paraffin Block RNA Isolation Kit (Ambion, Inc.) and RNeasyTM Mini kit (Qiagen, Chatsworth, Calif.).

[0181] The use of FFPE tissues as a source of RNA for RT-PCR has been described previously (Stanta et al., Biotechniques 11:304-308 (1991); Stanta el al., Methods Mol. Biol. 86:23-26 (1998); Jackson et al., Lancet 1:1391 (1989); Jackson et al., J. Clin. Pathol. 43:499-504 (1999); Finke et al., Biotechniques 14:448-453 (1993); Goldsworthy et al., Mol. Carcinog. 25:86-91 (1999); Stanta and Bonin, Biotechniques 24:271-276 (1998); Godfrey et al., J. Mol. Diagnostics 2:84 (2000); Specht el al., J. Mol. Med. 78:B27 (2000); Specht et al., Am. J. Pathol. 158:419-429 (2001)). For quick analysis of the RNA quality, RT-PCR can be performed utilizing a pair of primers targeting a short fragment in a highly expressed gene, for example, actin, ubiquitin, GAPDH or other well- described commonly used housekeeping gene. If the cDNA synthesized from the RNA sample can be amplified using this pair of primers, then the sample is suitable for the a quantitative measurements of RNA target sequences by any method preferred, for example, the DASL assay, which requires only a short cDNA fragment for the annealing of query oligonucleotides.

[0182] There are numerous tissue banks and collections including exhaustive samples from all stages of a wide variety of disease states, most notably cancer and in particular, pancreatic cancer. The ability to perform genotyping and / or gene expression analysis, including both qualitative and quantitative analysis on these samples enables the application of this methodology to the methods of the invention. In particular, the ability to establish a correlation of gene expression and a known predictor of disease extent and / or outcome by probing the genetic state of tissue samples for which clinical outcome is already known, allows for the establishment of a correlation between a particular molecular signature and the known predictor, such as a Gleason score, to derive a score that allows for a more sensitive prognosis than that based on the known predictor alone. The skilled person will appreciate that by building databases of molecular signatures from tissue samples of known outcomes, many such correlations can be established, thus allowing both diagnosis and prognosis of any condition. Thus, such approaches may be used to correlate the expression levels of the biomarkers of the invention.

[0183] Tissue samples useful for preparing a model for pancreatic cancer prediction include, for example, paraffin and polymer embedded samples, ethanol embedded samples and / or formalin and formaldehyde embedded tissues, although any suitable sample may be used. In general, nucleic acids isolated from archived samples can be highly degraded and the quality of nucleic preparation can depend on several factors, including the sample shelf life, fixation technique and isolation method. However, using the methodologies taught in U.S. Publ. No. 2004 / 0259105, which have the significant advantage that short or degraded targets can be used for analysis as long as the sequence is long enough to hybridize with the oligonucleotide probes, highly reproducible results can be obtained that closely mimic results found in fresh samples.

[0184] Archived tissue samples, which can be used for all methods of the invention, typically have been obtained from a source and preserved. Preferred methods of preservation include, but arc not limited to paraffin embedding, ethanol fixation and formalin, including formaldehyde and other derivatives, fixation as are known in the art. A tissue sample may be temporally "old", e.g. months or years old, or recently fixed. For example, post-surgical procedures generally include a fixation step on excised tissue for histological analysis. In a preferred embodiment, the tissue sample is a diseased tissue sample, particularly a pancreatic cancer tissue, including primary and secondary tumor tissues as well as lymph node tissue and metastatic tissue.

[0185] Thus, an archived sample can be heterogeneous and encompass more than one cell or tissue type, for example, tumor and non-tumor tissue. Generally, the invention methods can be practiced with the signature gene sequence contained in an archived sample or can be practiced with signature gene sequences that have been physically separated from the sample prior to performing a method of the invention.E. DETECTION AND / OR MEASUREMENT OF BIOMARKERS

[0186] The present invention contemplates any suitable means, techniques, and / or procedures for detecting and / or measuring the biomarkers of the invention. The skilled artisan will appreciate that the methodologies employed to measure the biomarkers of the invention will depend at least on the type of biomarker being detected or measured (e.g., lipid or polypeptide biomarker) and the source of the biological sample (e.g., whole bloodversus biopsy tissue). Certain biological samples may also require certain specialized treatments prior to measuring the biomarkers of the invention, e.g., the extraction of lipids from a serum in the case of lipid markers being measured. / . DETECTION OF PROTEIN MARKERS

[0187] The present invention contemplates any suitable method for detecting polypeptide biomarkers of the invention, i.e., SERPINA5, Vitronectin, Lumican, and Proteosome 20S subunit alpha 4. In certain embodiments, the detection method is an immunodetection method involving an antibody that specifically binds to one or more of the proteins from the group consisting of SERPINA5, Vitronectin, Lumican, and Proteosome 20S subunit alpha 4. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987), which is incorporated herein by reference.

[0188] In general, the immunobinding methods include obtaining a sample suspected of containing a biomarker protein, peptide or antibody, and contacting the sample with an antibody or protein or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[0189] The immunobinding methods include methods for detecting or quantifying the amount of a reactive component in a sample, which methods require the detection or quantitation of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing a pancreatic specific protein, peptide or a corresponding antibody, and contact the sample with an antibody or encoded protein or peptide, as the case may be, and then detect or quantify the amount of immune complexes formed under the specific conditions.

[0190] In terms of bio marker detection, the biological sample analyzed may be any sample that is suspected of containing one or more proteins from the group consisting of SERPINA5, Vitronectin, Lumican, and Proteosome 20S subunit alpha 4. The biological sample may be, for example, a pancreatic section or specimen, a homogenized tissue extract, an isolated cell, a cell membrane preparation, separated or purified forms of any of the above protein-containing compositions, or even any biological fluid that comesinto contact with pancreatic tissues, including blood or lymphatic fluid (e.g., plasma or buffy coat samples).

[0191] Contacting the chosen biological sample with the protein under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes). Generally, complex formation is a matter of simply adding the composition to the biological sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0192] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods arc generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. U.S. patents concerning the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin / avidin ligand binding arrangement, as is known in the art.

[0193] The protein employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.

[0194] Alternatively, the first added component that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the encoded protein, peptide or corresponding antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are thengenerally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0195] Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the encoded protein, peptide or corresponding antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[0196] The immunodetection methods of the present invention have evident utility in the prognosis of response to a drug treatment. Here, a biological or clinical sample suspected of containing either the encoded protein or peptide or corresponding antibody is used. However, these embodiments also have applications to non-clinical samples, such as in the tittering of antigen or antibody samples, in the selection of hybridomas, and the like.

[0197] The present invention, in particular, contemplates the use of ELISAs as a type of immunodetection assay. It is contemplated that the biomarker proteins or peptides of the invention will find utility as immunogens in ELISA assays in prognostic and monitoring response to a drug treatment. Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like also may be used.

[0198] In one exemplary ELISA, antibodies binding to the biomarkers of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the marker antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected.Detection is generally achieved by the addition of a second antibody specific for the target protein, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection also may be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0199] In another exemplary ELISA, the samples suspected of containing the marker of cancer subjects responsive to a drug treatment are immobilized onto the well surface and then contacted with the anti-biomarker antibodies of the invention. After binding and washing to remove non- specifically bound immune complexes, the bound antigen is detected. Where the initial antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.

[0200] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non- specifically bound species, and detecting the bound immune complexes. These are described as follows.

[0201] In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0202] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control human pancreatic, cancer and / or clinical or biological sample to be tested under conditions effective to allow immune complex (antigen / antibody) formation. Detection of theimmune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

[0203] The phrase "under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)ZTween. These added agents also tend to assist in the reduction of nonspecific background.

[0204] The "suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 h, at temperatures preferably on the order of 25 to 27°C, or may be overnight at about 4°C or so.

[0205] Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

[0206] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 h at room temperature in a PBS- containing solution such as PBS -Tween).

[0207] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.

[0208] The protein biomarkers of the invention can also be measured, quantitated, detected, and otherwise analyzed using protein mass spectrometry methods and instrumentation. Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Although not intending to be limiting, two approaches are typically used for characterizing proteins using mass spectrometry. In the first, intact proteins are ionized and then introduced to a mass analyzer. This approach is referred to as "top-down" strategy of protein analysis. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In the second approach, proteins are enzymatically digested into smaller peptides using a protease such as trypsin. Subsequently these peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Hence, this latter approach (also called "bottom-up" proteomics) uses identification at the peptide level to infer the existence of proteins.

[0209] Whole protein mass analysis of the biomarkers of the invention can be conducted using time-of-flight (TOE) MS, or Fourier transform ion cyclotron resonance (FT-ICR). These two types of instruments are useful because of their wide mass range, and in the case of FT-ICR, its high mass accuracy. The most widely used instruments for peptide mass analysis are the MAEDI time-of-flight instruments as they permit the acquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF can be analyzed in approx. 10 sec). Multiple stage quadrupole-time-of- flight and the quadrupole ion trap also find use in this application.

[0210] The protein biomarkers of the invention can also be measured in complex mixtures of proteins and molecules that co-exist in a biological medium or sample, however, fractionation of the sample may be required and is contemplated herein. It will be appreciated that ionization of complex mixtures of proteins can result in situation where the more abundant proteins have a tendency to “drown” or suppress signals from less abundant proteins in the same sample. In addition, the mass spectrum from a complex mixture can be difficult to interpret because of the overwhelming number of mixture components. Fractionation can be used to first separate any complex mixture of proteins prior to mass spectrometry analysis. Two methods are widely used to fractionateproteins, or their peptide products from an enzymatic digestion. The first method fractionates whole proteins and is called two-dimensional gel electrophoresis. The second method, high performance liquid chromatography (LC or HPLC) is used to fractionate peptides after enzymatic digestion. In some situations, it may be desirable to combine both of these techniques. Any other suitable methods known in the art for fractionating protein mixtures are also contemplated herein.

[0211] Gel spots identified on a 2D Gel are usually attributable to one protein. If the identity of the protein is desired, usually the method of in-gel digestion is applied, where the protein spot of interest is excised, and digested proteolytically. The peptide masses resulting from the digestion can be determined by mass spectrometry using peptide mass fingerprinting. If this infoimation does not allow unequivocal identification of the protein, its peptides can be subject to tandem mass spectrometry for de novo sequencing.

[0212] Characterization of protein mixtures using HPLC / MS may also be referred to in the art as “shotgun proteomics” and MuDPIT (Multi-Dimensional Protein Identification Technology). A peptide mixture that results from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography (LC). The eluent from the chromatography stage can be either directly introduced to the mass spectrometer through electrospray ionization, or laid down on a series of small spots for later mass analysis using MALDI.

[0213] The protein biomarkers of the present invention can be identified using MS using a variety of techniques, all of which are contemplated herein. Peptide mass fingerprinting uses the masses of proteolytic peptides as input to a search of a database of predicted masses that would arise from digestion of a list of known proteins. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample. It will be further appreciated that the development of methods and instrumentation for automated, data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS / MS) in conjunction with microcapillary liquid chromatography (LC) and database searching has significantly increased the sensitivity and speed of the identification of gel-separated proteins. Microcapillary LC-MS / MS has been usedsuccessfully for the large-scale identification of individual proteins directly from mixtures without gel electrophoretic separation (Link et al., 1999; Opitek et al., 1997).

[0214] Several recent methods allow for the quantitation of proteins by mass spectrometry. For example, stable (e.g., non-radioactive) heavier isotopes of carbon (13C) or nitrogen (15N) can be incorporated into one sample while the other one can be labeled with corresponding light isotopes (e.g. 12C and 14N). The two samples are mixed before the analysis. Peptides derived from the different samples can be distinguished due to their mass difference. The ratio of their peak intensities corresponds to the relative abundance ratio of the peptides (and proteins). The most popular methods for isotope labeling are SILAC (stable isotope labeling by amino acids in cell culture), trypsin-catalyzed 180 labeling, ICAT (isotope coded affinity tagging), iTRAQ (isobaric tags for relative and absolute quantitation). “Semi-quantitative” mass spectrometry can be performed without labeling of samples. Typically, this is done with MALDI analysis (in linear mode). The peak intensity, or the peak area, from individual molecules (typically proteins) is here correlated to the amount of protein in the sample. However, the individual signal depends on the primary structure of the protein, on the complexity of the sample, and on the settings of the instrument. Other types of "label-free" quantitative mass spectrometry, uses the spectral counts (or peptide counts) of digested proteins as a means for determining relative protein amounts.

[0215] In one embodiment, any one or more of the protein markers of the invention can be identified and quantified from a complex biological sample using mass spectroscopy in accordance with the following exemplary method, which is not intended to limit the invention or the use of other mass spectrometry-based methods.

[0216] In the first step of this embodiment, (A) a biological sample, e.g., a biological sample from a subject having cancer, which comprises a complex mixture of protein (including at least one biomarker of interest) is fragmented and labeled with a stable isotope X. (B) Next, a known amount of an internal standard is added to the biological sample, wherein the internal standard is prepared by fragmenting a standard protein that is identical to the at least one target biomarker of interest, and labeled with a stable isotope Y. (C) This sample obtained is then introduced in an LC-MS / MS device, and multiple reaction monitoring (MRM) analysis is performed using MRM transitionsselected for the internal standard to obtain an MRM chromatogram. (D) The MRM chromatogram is then viewed to identify a target peptide biomarker derived from the biological sample that shows the same retention time as a peptide derived from the internal standard (an internal standard peptide), and quantifying the target protein biomarker in the test sample by comparing the peak area of the internal standard peptide with the peak area of the target peptide biomarker.

[0217] Any suitable biological sample may be used as a starting point for LC- MS / MS / MRM analysis, including biological samples derived blood, urine, saliva, hair, cells, cell tissues, biopsy materials, and treated products thereof; and protein-containing samples prepared by gene recombination techniques.

[0218] Each of the above steps (A) to (D) is described further below.

[0219] Step (A) (Fragmentation and Labeling). In step (A), the target protein biomarker is fragmented to a collection of peptides, which is subsequently labeled with a stable isotope X. To fragment the target protein, for example, methods of digesting the target protein with a proteolytic enzyme (protease) such as trypsin, and chemical cleavage methods, such as a method using cyanogen bromide, can be used. Digestion by protease is preferable. It is known that a given mole quantity of protein produces the same mole quantity for each tryptic peptide cleavage product if the proteolytic digest is allowed to proceed to completion. Thus, determining the mole quantity of tryptic peptide to a given protein allows determination of the mole quantity of the original protein in the sample. Absolute quantification of the target protein can be accomplished by determining the absolute amount of the target protein-derived peptides contained in the protease digestion (collection of peptides). Accordingly, in order to allow the proteolytic digest to proceed to completion, reduction and alkylation treatments are preferably performed before protease digestion with trypsin to reduce and alkylate the disulfide bonds contained in the target protein.

[0220] Subsequently, the obtained digest (collection of peptides, comprising peptides of the target biomarker in the biological sample) is subjected to labeling with a stable isotope X. Examples of stable isotopes X include 1H and 2H for hydrogen atoms, 12C and 13C for carbon atoms, and 14N and 15N for nitrogen atoms. Any isotope can be suitably selected therefrom. Labeling by a stable isotope X can be performed by reactingthe digest (collection of peptides) with a reagent containing the stable isotope. Preferable examples of such reagents that are commercially available include mTRAQ (registered trademark) (produced by Applied Biosystems), which is an amine- specific stable isotope reagent kit. mTRAQ is composed of 2 or 3 types of reagents (mTRAQ-light and mTRAQ-heavy; or mTRAQ-DO, mTRAQ-D4, and mTRAQ-D8) that have a constant mass difference therebetween as a result of isotope-labeling, and that are bound to the N- terminus of a peptide or the primary amine of a lysine residue.

[0221] Step (B) (Addition of the Internal Standard). In step (B), a known amount of an internal standard is added to the sample obtained in step (A). The internal standard used herein is a digest (collection of peptides) obtained by fragmenting a protein (standard protein) consisting of the same amino acid sequence as the target protein (target biomarker) to be measured, and labeling the obtained digest (collection of peptides) with a stable isotope Y. The fragmentation treatment can be performed in the same manner as above for the target protein. Labeling with a stable isotope Y can also be performed in the same manner as above for the target protein. However, the stable isotope Y used herein must be an isotope that has a mass different from that of the stable isotope X used for labeling the target protein digest. For example, in the case of using the aforementioned mTRAQ (registered trademark) (produced by Applied Biosystems), when mTRAQ-light is used to label a target protein digest, mTRAQ-heavy should be used to label a standard protein digest.

[0222] Step (C) (LC-MS / MS and MRM Analysis). In step (C), the sample obtained in step (B) is first placed in an LC-MS / MS device, and then multiple reaction monitoring (MRM) analysis is performed using MRM transitions selected for the internal standard. By LC (liquid chromatography) using the LC-MS / MS device, the sample (collection of peptides labeled with a stable isotope) obtained in step (B) is separated first by onedimensional or multi-dimensional high-performance liquid chromatography. Specific examples of such liquid chromatography include cation exchange chromatography, in which separation is conducted by utilizing electric charge difference between peptides; and reversed-phase chromatography, in which separation is conducted by utilizing hydrophobicity difference between peptides. Both of these methods may be used in combination.

[0223] Subsequently, each of the separated peptides is subjected to tandem mass spectrometry by using a tandem mass spectrometer (MS / MS spectrometer) comprising two mass spectrometers connected in series. The use of such a mass spectrometer enables the detection of several fmol levels of a target protein. Furthermore, MS / MS analysis enables the analysis of internal sequence information on peptides, thus enabling identification without false positives. Other types of MS analyzers may also be used, including magnetic sector mass spectrometers (Sector MS), quadrupole mass spectrometers (QMS), time-of-flight mass spectrometers (TOFMS), and Fourier transform ion cyclotron resonance mass spectrometers (FT-ICRMS), and combinations of these analyzers.

[0224] Subsequently, the obtained data are put through a search engine to perform a spectral assignment and to list the peptides experimentally detected for each protein. The detected peptides arc preferably grouped for each protein, and preferably at least three fragments having an m / z value larger than that of the precursor ion and at least three fragments with an m / z value of, preferably, 500 or more are selected from each MS / MS spectrum in descending order of signal strength on the spectrum. From these, two or more fragments are selected in descending order of strength, and the average of the strength is defined as the expected sensitivity of the MRR transitions. When a plurality of peptides is detected from one protein, at least two peptides with the highest sensitivity are selected as standard peptides using the expected sensitivity as an index.

[0225] Step (D) (Quantification of the Target Protein in the Test Sample). Step (D) comprises identifying, in the MRM chromatogram detected in step (C), a peptide derived from the target protein (a target biomarker of interest) that shows the same retention time as a peptide derived from the internal standard (an internal standard peptide), and quantifying the target protein in the test sample by comparing the peak area of the internal standard peptide with the peak area of the target peptide. The target protein can be quantified by utilizing a calibration curve of the standard protein prepared beforehand.

[0226] The calibration curve can be prepared by the following method. First, a recombinant protein consisting of an amino acid sequence that is identical to that of the target biomarker protein is digested with a protease such as trypsin, as described above. Subsequently, precursor-fragment transition selection standards (PFTS) of a knownconcentration are individually labeled with two different types of stable isotopes (i.e.. one is labeled with a stable isomer used to label an internal standard peptide (labeled with IS), whereas the other is labeled with a stable isomer used to label a target peptide (labeled with T). A plurality of samples are produced by blending a certain amount of the IS- labeled PTFS with various concentrations of the T-labeled PTFS. These samples are placed in the aforementioned LC-MS / MS device to perform MRM analysis. The area ratio of the T-labeled PTFS to the IS-labeled PTFS (T-labeled PTFS / IS-labeled PTFS) on the obtained MRM chromatogram is plotted against the amount of the T-labeled PTFS to prepare a calibration curve. The absolute amount of the target protein contained in the test sample can be calculated by reference to the calibration curve.2. DETECTION OE NUCEEIC A CIDS CORRESPONDING TO PROTEINMARKERS

[0227] In certain embodiments, the invention involves the detection of nucleic acid biomarkers, e.g., the corresponding genes or mRNA of the protein markers of the invention, e.g., SERPINA5, Vitronectin, Lumican, and Proteosome 20S subunit alpha 4.

[0228] In various embodiments, the prognostic methods of the present invention generally involve the determination of expression levels of a set of genes in a biological sample. Determination of gene expression levels in the practice of the inventive methods may be performed by any suitable method. For example, determination of gene expression levels may be performed by detecting the expression of mRNA expressed from the genes of interest and / or by detecting the expression of a polypeptide encoded by the genes.

[0229] For detecting nucleic acids encoding biomarkers of the invention, any suitable method can be used, including, but not limited to, Southern blot analysis, Northern blot analysis, polymerase chain reaction (PCR) (see, for example, U.S. Pat. Nos. 4,683,195; 4,683,202, and 6,040,166; "PCR Protocols; A Guide to Methods and Applications", Innis et al. (Eds), 1990, Academic Press: New York), reverse transcriptase PCR (RT-PCT), anchored PCR, competitive PCR (see, for example, U.S. Pat. No. 5,747,251), rapid amplification of cDNA ends (RACE) (see, for example, "Gene Cloning and Analysis: Current Innovations, 1997, pp. 99- 115); ligase chain reaction (LCR) (see, for example,EP 01 320 308), one-sided PCR (Ohara et al., Proc. Natl. Acad. Sci., 1989, 86: 5673- 5677), in situ hybridization, Taqman-based assays (Holland et al., Proc. Natl. Acad. Sci., 1991, 88: 7276-7280), differential display (see, for example, Liang et al., Nucl. Acid. Res., 1993, 21: 3269-3275) and other RNA fingerprinting techniques, nucleic acid sequence based amplification (NASBA) and other transcription based amplification systems (see, for example, U.S. Pat. Nos. 5,409,818 and 5,554,527), Qbeta Replicase, Strand Displacement Amplification (SDA), Repair Chain Reaction (RCR), nuclease protection assays, subtraction-based methods, Rapid-Scan®, etc.

[0230] In other embodiments, gene expression levels of biomarkers of interest may be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyzing it using a microarray. A number of different array configurations and methods of their production are known to those skilled in the art (sec, for example, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637). Microarray technology allows for the measurement of the steady-state mRNA level of a large number of genes simultaneously. Microarrays currently in wide use include cDNA arrays and oligonucleotide arrays. Analyses using microarrays are generally based on measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid probe immobilized at a known location on the microarray (see, for example, U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and 6,271,002). Array-based gene expression methods are known in the art and have been described in numerous scientific publications as well as in patents (see, for example, M. Schena et al. , Science, 1995, 270: 467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA 1996, 93: 10614- 10619; J. J. Chen et al., Genomics, 1998, 51: 313-324; U.S. Pat. Nos. 5,143,854; 5,445,934; 5,807,522; 5,837,832; 6,040,138; 6,045,996; 6,284,460; and 6,607,885).

[0231] Nucleic acid used as a template for amplification can be isolated from cells contained in the biological sample, according to standard methodologies. (Sambrook et al., 1989) The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA. Inone embodiment, the RNA is whole cell RNA and is used directly as the template for amplification.

[0232] Pairs of primers that selectively hybridize to nucleic acids corresponding to any of the drug treatment responsive biomarker nucleotide sequences identified herein are contacted with the isolated nucleic acid under conditions that permit selective hybridization. Once hybridized, the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced. Next, the amplification product is detected. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax technology; Bcllus, 1994). Following detection, one may compare the results seen in a given patient with a statistically significant reference group of normal patients and cancer patients. In this way, it is possible to correlate the amount of nucleic acid detected with various clinical states.

[0233] The term primer, as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences may be employed. Primers may be provided in double- stranded or single- stranded form, although the single-stranded form is preferred.

[0234] A number of template dependent processes are available to amplify the nucleic acid sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each of which is incorporated herein by reference in its entirety.

[0235] In PCR, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target nucleic acid sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the target nucleic acid sequence is present in asample, the primers will bind to the target nucleic acid and the polymerase will cause the primers to be extended along the target nucleic acid sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target nucleic acid to form reaction products, excess primers will bind to the target nucleic acid and to the reaction products and the process is repeated.

[0236] A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook el al., 1989. Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90 / 07641 filed Dec. 21, 1990. Polymerase chain reaction methodologies are well known in the art.

[0237] Another method for amplification is the ligase chain reaction ("LCR"), disclosed in European Application No. 320 308, incorporated herein by reference in its entirely. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar- to LCR for binding probe pairs to a target sequence.

[0238] Qbeta Replicase, described in PCT Application No. PCT / US87 / 00880, also may be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA which has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which may then be detected.

[0239] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'- [a-thio] -triphosphates in one strand of a restriction site also may be useful in the amplification of nucleic acids in the present invention. Walker et al. (1992), incorporated herein by reference in its entirety.

[0240] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases may be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences also may be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.

[0021] Still other amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT / US89 / 01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and / or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

[0242] Other contemplated nucleic acid amplification procedures include transcriptionbased amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR. Kwoh et al. (1989); Gingeras et al., PCT Application WO 88 / 10315, incorporated herein by reference in their entirety. In NASBA, the nucleic acids may be prepared for amplification by standard phenol / chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA / RNA hybrids are digested with RNase H while double strandedDNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into double stranded DNA, and transcribed once against with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

[0243] Davey el al., European Application No. 329 822 (incorporated herein by reference in its entirely) disclose a nucleic acid amplification process involving cyclically synthesizing single- stranded RNA ("ssRNA"), ssDNA, and double- stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dcpcndcnt DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H(RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase 1), resulting in a double- stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence may be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies may then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification may be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence may be chosen to be in the form of either DNA or RNA.

[0244] Miller et al., PCT Application WO 89 / 06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter / primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Otheramplification methods include "race" and "one-sided PCR." Frohman (1990) and Ohara et al. (1989), each herein incorporated by reference in their entirety.

[0245] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, also may be used in the amplification step of the present invention. Wu et al. (1989), incorporated herein by reference in its entirety.

[0246] Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular- needs and targeted sequences employed. In a preferred embodiment, the oligonucleotide probes or primers are at least 10 nucleotides in length (preferably, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 . . . ) and they may be adapted to be especially suited for a chosen nucleic acid amplification system and / or hybridization system used. Longer probes and primers arc also within the scope of the present invention as well known in the art. Primers having more than 30, more than 40, more than 50 nucleotides and probes having more than 100, more than 200, more than 300, more than 500 more than 800 and more than 1000 nucleotides in length are also covered by the present invention. Of course, longer primers have the disadvantage of being more expensive and thus, primers having between 12 and 30 nucleotides in length are usually designed and used in the art. As well known in the art, probes ranging from 10 to more than 2000 nucleotides in length can be used in the methods of the present invention. As for the % of identity described above, non- specific ally described sizes of probes and primers (e.g., 16, 17, 31, 24, 39, 350, 450, 550, 900, 1240 nucleotides, . . . ) are also within the scope of the present invention. In one embodiment, the oligonucleotide probes or primers of the present invention specifically hybridize with a marker RNA (or its complementary sequence) or a marker mRNA. More preferably, the marker primers and probes will be chosen to detect a marker RNA which is associated with subjects responsive to a drug treatment.

[0247] In other embodiments, the detection means can utilize a hybridization technique, e.g., where a specific primer or probe is selected to anneal to a target biomarker of interest and thereafter detection of selective hybridization is made. As commonly known in the art, the oligonucleotide probes and primers can be designed by taking intoconsideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning— A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1994, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).

[0248] To enable hybridization to occur under the assay conditions of the present invention, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%, 74%), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) and more preferably at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to a portion of a filamin A or polynucleotide of another biomarker of the invention. Probes and primers of the present invention are those that hybridize under stringent hybridization conditions and those that hybridize to biomarker homologs of the invention under at least moderately stringent conditions. In certain embodiments probes and primers of the present invention have complete sequence identity to the biomarkers of the invention (e.g. calbindin 2, gene sequences (e.g., cDNA or mRNA). It should be understood that other probes and primers could be easily designed and used in the present invention based on the biomarkers of the invention disclosed herein by using methods of computer alignment and sequence analysis known in the art (cf. Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000).3. ANTIBODIES AND LABELS

[0249] In some embodiments, the invention provides methods and compositions that include labels for the highly sensitive detection and quantitation of the markers of the invention. One skilled in the art will recognize that many strategies can be used for labeling target molecules to enable their detection or discrimination in a mixture of particles. The labels may be attached by any known means, including methods that utilize non-specific or specific interactions of label and target. Labels may provide a detectable signal or affect the mobility of the particle in an electric field. In addition, labeling can be accomplished directly or through binding partners.

[0250] In some embodiments, the label comprises a binding partner that binds to the biomarker of interest, where the binding partner is attached to a fluorescent moiety. The compositions and methods of the invention may utilize highly fluorescent moieties, e.g., a moiety capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. Moieties suitable for the compositions and methods of the invention are described in more detail below.

[0251] In some embodiments, the invention provides a label for detecting a biological molecule comprising a binding partner for the biological molecule that is attached to a fluorescent moiety, wherein the fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. In some embodiments, the moiety comprises a plurality of fluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 fluorescent entities. In some embodiments, the moiety comprises about 2 to 4 fluorescent entities. In some embodiments, the biological molecule is a protein or a small molecule. In some embodiments, the biological molecule is a protein. The fluorescent entities can be fluorescent dye molecules. In some embodiments, the fluorescent dye molecules comprise at least one substituted indolium ring system in which the substituent on the 3- carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In some embodiments, the dye molecules are Alexa Fluor molecules selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. hi some embodiments, the dye molecules are Alexa Fluor molecules selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the dye molecules are Alexa Fluor 647 dye molecules. In some embodiments, the dye molecules comprise a first type and a second type of dye molecules, e.g., two different Alexa Fluor molecules, e.g., where thefirst type and second type of dye molecules have different emission spectra. The ratio of the number of first type to second type of dye molecule can be, e.g., 4 to 1, 3 to 1, 2 to 1, 1 to 1, 1 to 2, 1 to 3 or 1 to 4. The binding partner can be, e.g., an antibody.

[0252] In some embodiments, the invention provides a label for the detection of a biological marker of the invention, wherein the label comprises a binding partner for the marker and a fluorescent moiety, wherein the fluorescent moiety is capable of emitting at least about 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules. In some embodiments, the fluorescent moiety comprises a fluorescent molecule, hi some embodiments, the fluorescent moiety comprises a plurality of fluorescent molecules, e.g., about 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, or 3 to 6 fluorescent molecules. In some embodiments, the label comprises about 2 to 4 fluorescent molecules. In some embodiments, the fluorescent dye molecules comprise at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In some embodiments, the fluorescent molecules are selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the fluorescent molecules are selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the fluorescent molecules are Alexa Fluor 647 molecules. In some embodiments, the binding partner comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0253] The term "antibody," as used herein, is a broad term and is used in its ordinary sense, including, without limitation, to refer to naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. An "antigen-binding fragment" of an antibody refers to the part of the antibody that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L")chains. It will be appreciated that the choice of epitope or region of the molecule to which the antibody is raised will determine its specificity, e.g., for various forms of the molecule, if present, or for total (e.g., all, or substantially all of the molecule).

[0254] Methods for producing antibodies are well-established. One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art will also appreciate that binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). Monoclonal and polyclonal antibodies to molecules, e.g., proteins, and markers also commercially available (R and D Systems, Minneapolis, Minn.; HyTcst, HyTcst Ltd., Turku Finland; Abeam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049 USA; BiosPacific, Emeryville, Calif.).

[0255] In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody.

[0256] Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.

[0257] Monoclonal antibodies may be prepared using hybridoma methods, such as the technique of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976), and improvements thereto. These methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity. Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding antibodies employed in the disclosed methods may be isolated and sequenced using conventional procedures. Recombinant antibodies, antibody fragments, and / or fusions thereof, can be expressed in vitro or in prokaryotic cells (e.g. bacteria) oreukaryotic cells (e.g. yeast, insect or mammalian cells) and further purified as necessary using well known methods.

[0258] More particularly, monoclonal antibodies (MAbs) may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified expressed protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB / c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

[0259] The animals are injected with antigen as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two- week intervals. Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.

[0260] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency,and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

[0261] The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones may then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma may be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, may then be tapped to provide MAbs in high concentration. The individual cell lines also may be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they may be readily obtained in high concentrations. MAbs produced by cither means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

[0262] Large amounts of the monoclonal antibodies of the present invention also may be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into mammals which are histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.

[0263] In accordance with the present invention, fragments of the monoclonal antibody of the invention may be obtained from the monoclonal antibody produced as described above, by methods which include digestion with enzymes such as pepsin or papain and / or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention may be synthesized using an automated peptide synthesizer.

[0264] Antibodies may also be derived from a recombinant antibody library that is based on amino acid sequences that have been designed in silico and encoded by polynucleotides that are synthetically generated. Methods for designing and obtaining in silico-created sequences are known in the art (Knappik et al., J. Mol. Biol. 296:254:57- 86, 2000; Krebs et al., J. Immunol. Methods 254:67-84, 2001 ; U.S. Pat. No. 6,300,064).

[0265] Digestion of antibodies to produce antigen -binding fragments thereof can be performed using techniques well known in the art. For example, the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the "F(ab)" fragments) each comprise a covalent heterodimer that includes an intact antigenbinding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the "F(ab')2" fragment, which comprises both antigen-binding sites. "Fv" fragments can be produced by preferential proteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, but are more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule (Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochcm. 19:4091-4096 (1980)).

[0266] Antibody fragments that specifically bind to the protein biomarkers disclosed herein can also be isolated from a library of scFvs using known techniques, such as those described in U.S. Pat. No. 5,885,793.

[0267] A wide variety of expression systems are available in the art for the production of antibody fragments, including Fab fragments, scFv, VL and VHs. For example, expression systems of both prokar yotic and eukaryotic origin may be used for the large- scale production of antibody fragments. Particularly advantageous are expression systems that permit the secretion of large amounts of antibody fragments into the culture medium. Eukaryotic expression systems for large-scale production of antibody fragments and antibody fusion proteins have been described that are based on mammalian cells, insect cells, plants, transgenic animals, and lower eukaryotes. For example, the cost-effective, large-scale production of antibody fragments can be achieved in yeast fermentation systems. Large-scale fermentation of these organisms is well known in the ail and is currently used for bulk production of several recombinant proteins.

[0268] Antibodies that bind to the protein biomarkers employed in the present methods are, in some cases, available commercially or can be obtained without undue experimentation.

[0269] In still other embodiments, particularly where oligonucleotides are used as binding partners to detect and hybridize to mRNA biomarkers or other nucleic acid based biomarkers, the binding partners (e.g., oligonucleotides) can comprise a label, e.g., a fluorescent moiety or dye. In addition, any binding partner of the invention, e.g., an antibody, can also be labeled with a fluorescent moiety. The fluorescence of the moiety will be sufficient to allow detection in a single molecule detector, such as the single molecule detectors described herein. A "fluorescent moiety," as that term is used herein, includes one or more fluorescent entities whose total fluorescence is such that the moiety may be detected in the single molecule detectors described herein. Thus, a fluorescent moiety may comprise a single entity (e.g., a Quantum Dot or fluorescent molecule) or a plurality of entities (e.g., a plurality of fluorescent molecules). It will be appreciated that when "moiety," as that term is used herein, refers to a group of fluorescent entities, e.g., a plurality of fluorescent dye molecules, each individual entity may be attached to the binding partner separately or the entities may be attached together, as long as the entities as a group provide sufficient fluorescence to be detected.

[0270] Typically, the fluorescence of the moiety involves a combination of quantum efficiency and lack of photobleaching sufficient that the moiety is detectable above background levels in a single molecule detector, with the consistency necessary for the desired limit of detection, accuracy, and precision of the assay. For example, in some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and / or quantitation of a molecule, e.g., a marker, at a limit of detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.00001, or 0.000001 pg / ml and with a coefficient of variation of less than about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less, e.g., about 10% or less, in the instruments described herein. In some embodiments, the fluorescence of the fluorescent moiety is such that it allows detection and / or quantitation of a molecule, e.g., a marker, at a limit of detection of less than about 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 pg / ml and with a coefficient of variation of less than about 10%, in the instruments described herein. "Limit of detection," or LoD, as those terms are used herein, includes the lowest concentration at which one can identify a sample as containing a molecule of the substance of interest, e.g., the first non-zero value. It can be defined by the variability of zeros and the slope of the standard curve. For example, thelimit of detection of an assay may be determined by running a standard curve, determining the standard curve zero value, and adding 2 standard deviations to that value. A concentration of the substance of interest that produces a signal equal to this value is the "lower limit of detection" concentration.

[0271] Furthermore, the moiety has properties that are consistent with its use in the assay of choice. In some embodiments, the assay is an immunoassay, where the fluorescent moiety is attached to an antibody; the moiety must have properties such that it does not aggregate with other antibodies or proteins, or experiences no more aggregation than is consistent with the required accuracy and precision of the assay. In some embodiments, fluorescent moieties that are preferred are fluorescent moieties, e.g., dye molecules that have a combination of 1) high absorption coefficient; 2) high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with labeling the molecule of interest e.g., protein) so that it may be analyzed using the analyzers and systems of the invention (e.g., does not cause precipitation of the protein of interest, or precipitation of a protein to which the moiety has been attached).

[0272] Any suitable fluorescent moiety may be used. Examples include, but are not limited to, Alexa Fluor dyes (Molecular Probes, Eugene, Oreg.). The Alexa Fluor dyes are disclosed in U.S. Pat. Nos. 6,977,305; 6,974,874; 6,130,101; and 6,974,305 which are herein incorporated by reference in their entirety. Some embodiments of the invention utilize a dye chosen from the group consisting of Alexa Fluor 647, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Some embodiments of the invention utilize a dye chosen from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Some embodiments of the invention utilize a dye chosen from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Some embodiments of the invention utilize the Alexa Fluor 647 molecule, which has an absorption maximum between about 650 and 660 nm and an emission maximum between about 660 and 670 nm. The Alexa Fluor 647 dye is used alone or in combination with other Alexa Fluor dyes.

[0273] In some embodiments, the fluorescent label moiety that is used to detect a biomarker in a sample using the analyzer systems of the invention is a quantum dot. Quantum dots (QDs), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals that contain anywhere between 100 to 1,000 electrons and range from 2-10 nm. Some QDs can be between 10-20 nm in diameter. QDs have high quantum yields, which makes them particularly useful for optical applications. QDs are fluorophores that fluoresce by forming excitons, which are similar to the excited state of traditional fluorophores, but have much longer lifetimes of up to 200 nanoseconds. This property provides QDs with low photobleaching. The energy level of QDs can be controlled by changing the size and shape of the QD, and the depth of the QDs' potential. One optical feature of small excitonic QDs is coloration, which is determined by the size of the dot. The larger the dot, the redder, or more towards the red end of the spectrum the fluorescence. The smaller the dot, the bluer or more towards the blue end it is. The bandgap energy that determines the energy and hence the color of the fluoresced light is inversely proportional to the square of the size of the QD. Larger QDs have more energy levels which are more closely spaced, thus allowing the QD to absorb photons containing less energy, i.e., those closer to the red end of the spectrum. Because the emission frequency of a dot is dependent on the bandgap, it is possible to control the output wavelength of a dot with extreme precision. In some embodiments the protein that is detected with the single molecule analyzer system is labeled with a QD. In some embodiments, the single molecule analyzer is used to detect a protein labeled with one QD and using a filter to allow for the detection of different proteins at different wavelengths.4. DETECTION OF LIPID AND METABOLITE MARKERS

[0274] The present invention contemplates any suitable methods for detecting lipid and metabolite biomarkers of the invention, i.e., 2-keto-isovalerate, PI- 18:0 / 22:4, PI- 18:0 / 20:2, PA-P18:0 / 18:0, SL-7-HDHA, and PC-38:8 / O-38:l.

[0275] A lipid sample may be extracted from a biological sample using any method known in the ait such as chloroform- methanol based methods, isopropanol-hexane methods, the Bligh & Dyer lipid extraction method or a modified version thereof, or anycombination thereof. Suitable modifications to the Bligh & Dyer method include treatment of crude lipid extracts with lithium methoxide followed by subsequent liquidliquid extraction to remove generated free fatty acids, fatty acid methyl esters, cholesterol, and water-soluble components that may hinder the shotgun analysis of sphingolipidomes. Since sphingolipids are inert to the described base-treatment, the global analysis and accurate quantitation to assess low and even very low abundant sphingolipids is possible by using a modified Bligh & Dyer method. Following lipid extraction, it may be beneficial to separate the lipids prior to mass spectro metric analysis. Methods for separating lipids are known in the art. Suitable methods include, but are not limited to, chromatography methods such as solid-phase extraction, high performance liquid chromatography (HPLC), normal-phase HPLC, or reverse-phase HPLC. The resultant lipid extracts are then analyzed by mass spectrometric techniques commonly known in the art.

[0276] Detection and measurement of metabolites may be carried out using techniques commonly known in the art. For example, metabolomics analysis is described in Tolstikov V, Nikolayev A, Dong S, Zhao G, Kuo MS. Metabolomics Analysis of Metabolic Effects of Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibition on Human Cancer Cells. PLoS One. 2014;9:el 14019, the contents of which is hereby incorporated herein by reference. Exemplary separation protocols which can be used in metabolite analysis include GC-MS, LC-MS, GC-TOF-MS, HILIC-LC-MS / MS, and RP- LC-HRMS analyses.

[0277] Web based databases having high resolution MS data, for example METLIN (see the website metlin.scripps.edu / index.php), The Human Metabolome Database (HMDB) (see the website hmdb.ca / ), MASSBANK (see the website massbank.jp / ), NIST-MS (see the website chemdata.nist.gov / ), IDEOME (see the website mzmatch.sourceforge.net / ideom.php), mzCloud (see the website mzcloud.org / ) and other libraries can be used for the elemental composition assignment, spectral data comparisons, and detailed manual interpretation.F. PROGNOSTIC AND TREATMENT METHODS

[0278] The invention provides methods for prognosing a response to Coenzyme Q10 treatment for pancreatic cancer in a subject, comprising:(a) detecting the level of one or more markers in a biological sample from the subject, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA- P18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l; and(b) comparing the level of the one or more markers in the biological sample with a predetermined threshold value; wherein an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PL18:0 / 22:4, PI-18:0 / 20:2, and / or PA-P18:O / 18:O relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q 10 treatment; and / or wherein a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

[0279] The invention also provides methods for prognosing a response to Coenzyme Q10 treatment in a subject, wherein the prognosis is determined concurrently with the diagnosis of pancreatic cancer in the subject, comprising:(a) diagnosing the subject with pancreatic cancer;(b) detecting the level of one or more markers in a biological sample from the subject, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1; and(c) comparing the level of the one or more markers in the biological sample with a predetermined threshold value; wherein an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and / or PA-P18:O / 18:O relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment; and / orwherein a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

[0280] In some embodiments, an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, and / or PA-P 18:0 / 18:0 relative to the predetermined threshold value, and / or a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38: 8 / 0-38.1 relative to the predetermined threshold value indicates that the subject will exhibit stable disease in response to the Coenzyme Q10 treatment.

[0281] In some embodiments, the response to the Coenzyme Q10 treatment comprises no change or a decrease in tumor size. In other embodiments, the response to the Coenzyme Q10 treatment comprises an increase in overall days of survival.

[0282] In some embodiments, the method further comprises selecting the subject for administration of a treatment regimen comprising Coenzyme Q10 based on the prognosis for the Coenzyme Q10 treatment in the subject, z.e., selecting the subject for the treatment regimen comprising Coenzyme Q10 when there is an indication that the subject will be responsive to the Coenzyme Q10 treatment. In some embodiments, the method further comprises selecting and / or administering a treatment regimen based on the prediction of the Coenzyme Q10 treatment in the subject. In some embodiments, the treatment regimen comprises further monitoring the subject for progression of pancreatic cancer. In some embodiments, the treatment regimen is selected from the group consisting of (a) radiation therapy, (b) chemotherapy, (c) surgery, (d) hormone therapy, (e) antibody therapy, (f) immunotherapy, (g) cytokine therapy, (h) growth factor therapy, (i) watchful waiting, and (i) any combination of (a)-(i).

[0283] In some embodiments, the treatment regimen comprises administering Coenzyme Q10 to the subject. In some embodiments, the treatment regimen comprises administering Coenzyme Q10 in combination with administration of gemcitabine to the subject.

[0284] In some embodiments, the subject has been previously diagnosed with pancreatic cancer, optionally who has been treated previously for the pancreatic cancer (e.g., 1, 2, 3 or more times), and optionally has failed all of those treatments.

[0285] The invention further provides methods for detecting a marker useful for prognosing response to Coenzyme Q10 treatment for pancreatic cancer, comprising:(a) obtaining a sample from a subject diagnosed with pancreatic cancer; and(b) detecting the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1.

[0286] The invention also provides methods for treating pancreatic cancer in a subject comprising:(a) obtaining a biological sample from the subject;(b) submitting the biological sample to obtain prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-kcto- isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2; and(c) administering a therapeutically effective amount of Coenzyme Q10 to the subject if the level of the one or more markers is above or below a predetermined threshold value.

[0287] The invention further provides methods for treating pancreatic cancer in a subject comprising:(a) obtaining prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2; and(b) administering a therapeutically effective amount of Coenzyme Q10 to the subject if the level of the one or more markers is above or below a predetermined threshold value.

[0288] The invention also provides methods for treating pancreatic cancer in a subject suspected of having pancreatic cancer, the method comprising:(a) obtaining a biological sample from the subject for use in identifying prognostic information as to the level of one or more markers in the biological sample,wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2;(b) measuring the level of the one or more markers in the biological sample; and

[0289] (c) recommending to a healthcare provider to administer Coenzyme Q10 if the level of the one or more markers in the biological sample is above or below a predetermined threshold value.

[0290] In some embodiments, an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and / or PA-P18:O / 18:O relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment; and / or a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

[0291] In some embodiments, the Coenzyme Q10 treatment is administered by intravenous administration, optionally, the Coenzyme Q10 treatment is administered by continuous infusion. In some embodiments, the Coenzyme Q10 treatment is administered by continuous infusion for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours. In some embodiments, the Coenzyme Q10 treatment is administered by a regimen described in PCT / US2014 / 054155, US Patent No. 9 / 901,542, or US Patent No. 11 / 298,313, the entire contents of each of which are incorporated in their entirety by reference herein.

[0292] In some embodiments, the methods further comprise administering a therapeutically effective amount of a second drug. In some embodiments, the second drug is gemcitabine.

[0293] In some embodiments, the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers is determined.

[0294] In some embodiments, the level of the one or more markers is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof. In some embodiments, the level of the one or more markers is detected by determining the level of its corresponding mRNA in the biological sample. In some embodiments, the level of theone or more markers is detected by determining the level of its protein in the biological sample.

[0295] The invention also provides methods for monitoring the treatment of pancreatic cancer in a subject by(a) contacting a first biological sample obtained from the subject prior to administering at least a portion of a treatment regimen to the subject with a panel of one or more detection reagents wherein each detection reagent is specific for one or more markers, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l;(b) contacting a second biological sample obtained from the subject after administering at least a portion of a treatment regimen to the subject with a panel of one or more detection reagents wherein each detection reagent is specific for one or more markers, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l;(c) measuring the level of the one or more markers in the first biological sample and the second biological sample by each detection reagent; and(d) comparing the level of the one or more markers in the first sample with the level of one or more of markers in the second sample, thereby monitoring the treatment of pancreatic cancer in the subject.

[0296] The invention provides methods of selecting for administration of active treatment or against administration of active treatment of pancreatic cancer in a subject by(a) contacting a first biological sample obtained from the subject prior to administering a treatment regimen to the subject with a panel of one or more detection reagents wherein each detection reagent is specific one or more markers, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, Pl-18:0 / 22:4, Pl-18:0 / 20:2, PA-P 18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l;(b) contacting a second biological sample obtained from the subject after administering a treatment regimen to the subject with a panel of one or more detectionreagents wherein each detection reagent is specific one or more markers, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l;(c) measuring the level of the one or more markers detected in the first biological sample and the second biological sample by each detection reagent; and(d) comparing the level of the one or more markers in the first sample with the level of the one or more markers in the second sample, wherein selecting for administration of active treatment (e.g., treatment comprising administration of Coenzyme Q10) or against administration of active treatment of pancreatic cancer is based on the presence or absence of changes in the level of one or more markers between the first sample and the second sample.

[0297] In certain embodiments of the methods provided herein, the one or more markers are two or more markers. In certain embodiments of the methods provided herein, the one or more markers are three or more markers. In certain embodiments of the methods provided herein, the one or more markers are four or more markers. In certain embodiments of the methods provided herein, the one or more markers are five or more markers. In certain embodiments of the methods provided herein, the one or more markers are six or more markers. In certain embodiments of the methods provided herein, the one or more markers are seven or more markers. In certain embodiments of the methods provided herein, the one or more markers are eight or more markers. In certain embodiments of the methods provided herein, the one or more markers are nine or more markers.

[0298] In certain embodiments of the methods provided herein, an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and / or PA-P18:0 / 18:0 relative to the predetermined threshold value, and / or a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

[0299] In certain embodiments of the methods provided herein, an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PLl 8:0 / 22:4, PLl 8:0 / 20:2, and / orPA-P18:0 / 18:0 relative to the predetermined threshold value, and / or a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will exhibit stable disease in response to the Coenzyme Q10 treatment.

[0300] In any of the aforementioned embodiments, the methods may also include a step of determining whether a subject having pancreatic cancer or who is being treated for pancreatic cancer is responsive to a particular treatment (e.g., treatment comprising administration of Coenzyme Q10). Such a step can include, for example, measuring the level of one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l prior to administering a treatment, and measuring the level of expression of one or more markers after administering the treatment, and comparing the level of the markers before and after treatment. Determining that the subject is responsive to the treatment if the level of the one or more markers is different before treatment as compared to after treatment. The method may further include the step of adjusting the treatment to a higher dose (e.g., of Coenzyme Q10) in order to increase the responsiveness to the treatment, or adjusting the treatment to a lower dose in order to decrease the responsiveness to the treatment.

[0301] In certain embodiments, the methods provided herein further comprise comparing the detected level of the one or more markers in the biological samples with one or more control samples wherein the control sample is one or more of a sample from the same subject at an earlier time point than the biological sample.

[0302] Certain other embodiments of the methods further comprise determining the particular’ stage or grade of pancreatic cancer. In other embodiments, the present invention also involves the analysis and consideration of any clinical and / or patient- related health data, for example, data obtained from an Electronic Medical Record (e.g., collection of electronic health information about individual patients or populations relating to various types of data, such as, demographics, medical history, medication and allergies, immunization status, laboratory test results, radiology images, vital signs, personal statistics like age and weight, and billing information).

[0303] In certain embodiments the methods provided herein further comprising obtaining a subject sample. In some embodiments, the biological sample comprises a blood sample or a component thereof. In some embodiments, the sample comprises a buffy coat sample. In some embodiments, the sample comprises a plasma sample.

[0304] In certain embodiments the methods provided herein further comprise selecting a treatment regimen for the subject based on the level of one or more of the markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l.

[0305] In certain embodiments the methods provided herein further comprise selecting a subject for having or being suspected of having pancreatic cancer.

[0306] In certain embodiments the methods provided herein further comprising treating the subject with a regimen including one or more treatments as described herein.

[0307] In certain embodiments the methods provided herein further comprise selecting the one or more specific treatment regimens for the subject based on the results of the prognostic and monitoring methods provided herein. In one embodiment, a treatment regimen known to be effective against pancreatic cancer having the marker signature detected in the subject / sample is selected for the subject. In certain embodiments, the treatment method is started, change, revised, or maintained based on the results from the prognostic methods of the invention, e.g., when it is determined that the subject is responding to the treatment regimen, or when it is determined that the subject is not responding to the treatment regimen, or when it is determined that the subject is insufficiently responding to the treatment regimen. In certain embodiments, the treatment method is changed based on the results from the prognostic methods.

[0308] In certain other embodiments the methods provided herein further comprise introducing one or more specific treatment regimens e.g., treatment regimen that comprises administration of Coenzyme Q10) for the subject based on the results of the prognostic and monitoring methods provided herein. In one embodiment, a treatment regimen known to be effective against pancreatic cancer is selected for the subject. In certain embodiments, the treatment method is stalled, change, revised, or maintained based on the results from the prognostic methods of the invention, e.g., when it isdetermined that the subject is responding to the treatment regimen, or when it is determined that the subject is not responding to the treatment regimen, or when it is determined that the subject is insufficiently responding to the treatment regimen. In certain embodiments, the treatment method is changed based on the results from the prognostic methods.

[0309] In yet other embodiments the methods provided herein further comprise the step of administering a therapeutically effective amount of an anti-pancreatic cancer therapy (e.g., anti-pancreatic cancer therapy that comprises administration of Coenzyme Q10) based on the results of the prognostic and monitoring methods provided herein. In one embodiment, a treatment regimen known to be effective against pancreatic cancer is selected for the subject. In certain embodiments, the treatment method is administered based on the results from the prognostic or prognostic methods of the invention, e.g., when it is determined that the subject expresses one or more markers of the invention above or below some threshold level that is indicative of responsiveness to a treatment.

[0310] In yet other embodiments the methods provided herein further comprise the step of increasing, decreasing, or changing the dose of an anti-pancreatic cancer therapy (e.g., anti-pancreatic cancer therapy that comprises administration of Coenzyme Q10) based on the results of the prognostic and monitoring methods provided herein. In one embodiment, a treatment regimen known to be effective against pancreatic cancer is selected for the subject. In certain embodiments, the treatment method is administered based on the results from the prognostic methods of the invention, e.g., when it is determined that the subject expresses one or more markers of the invention above or below some threshold level that is indicative of responsiveness to a treatment.

[0311] In yet other embodiments the methods provided herein further comprise the step of increasing, decreasing, or changing the dose of an anti-pancreatic cancer therapy e.g., anti-pancreatic cancer therapy that comprises administration of Coenzyme Q10) based on the results of the prognostic and monitoring methods provided herein. In one embodiment, a treatment regimen known to be effective against pancreatic cancer is selected for the subject. In certain embodiments, the treatment method is administered based on the results from the prognostic or prognostic methods of the invention, e.g.,when it is determined that the subject expresses one or more markers of the invention above or below some threshold level that is indicative of responsiveness to a treatment.

[0312] In certain embodiments of the methods provided herein, the method further comprises isolating a component of the biological sample, for example a protein, a lipid, or a metabolite.

[0313] In certain embodiments of the methods provided herein, the method further comprises labeling a component of the biological sample, for example a protein, a lipid, or a metabolite.

[0314] In certain embodiments of the methods provided herein, the method further comprises amplifying a component of a biological sample, for example a nucleic acid.

[0315] In certain embodiments of the methods provided herein, the method comprises forming a complex with a probe and a component of a biological sample. In certain embodiments, forming a complex with a probe comprises forming a complex with at least one non-naturally occurring reagent. In certain embodiments of the methods provided herein, the method comprises processing the biological sample. In certain embodiments of the methods provided herein, the method of detecting a level of at least two markers comprises a panel of markers. In certain embodiments of the methods provided herein, the method of detecting a level comprises attaching the marker to be detected to a solid surface.MONITORING CLINICAL TRIALS

[0316] Monitoring the influence of agents (e.g.. drug compounds) on the level of a marker of the invention can be applied not only in basic drug screening or monitoring the treatment of a single subject, but also in clinical trials. For example, the effectiveness of an agent to affect marker levels can be monitored in clinical trials of subjects receiving treatment for pancreatic cancer. In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g.. an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) comprising the steps of (i) obtaining a preadministration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of one or more selected markers of the invention in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of the marker(s) in the post-administration samples; (v) comparing the level of the marker(s) in the pre-administration sample with the level of the marker(s) in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, an increase in the level of the marker during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage. In other embodiments, a decrease in the level of the marker during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage. Conversely, in some embodiments, a decrease in the level of the marker may indicate efficacious treatment and no need to change dosage. In other embodiments, an increase in the level of the marker may indicate efficacious treatment and no need to change dosage.G. COENZYME Q10

[0317] It will be understood that the methods provided herein are contemplated to be carried out with a composition comprising one or more Coenzyme Q10 compounds.Coenzyme Q10 compounds are intended to include a class of CoQlO compounds.Coenzyme Q10 compounds effective for the methods described herein include coenzyme Q10, a metabolite of coenzyme Q10, a biosynthetic precursor of coenzyme Q10, an analog of coenzyme Q10, a derivative of coenzyme Q10, and coenzyme Q10 related compounds. An analog of coenzyme Q10 includes analogs having no or at least one isoprenyl repeats. Coenzyme Q10 has the following structure:wherein x is 10. hi the instant invention, CoQlO compounds can include derivatives of coenzyme Q10 in which x is any number of isoprenyl units from 4-10, or any number ofisoprenyl units from 6-10, or any number of isoprenyl units from 8-10, or 9-10 isoprenyl units. Coenzyme Q10 includes the fully oxidized version, also known as ubiquinone, the partially oxidized version, also known as semiquinone or ubisemiquinone, or the fully reduced version, also known as ubiquinol; or any mixtures or combinations thereof. In certain embodiments, the CoQlO compound for treatment of cancer is ubiquinone. In certain embodiments, the CoQlO compound for treatment of cancer is ubiquinol.

[0318] In certain embodiments of the present invention, the therapeutic agent is coenzyme Q10 (CoQlO). Coenzyme Q10, also referred to herein as CoQlO, is also known as ubiquinone, or ubidecarenone. Coenzyme Q10 is art-recognized and further described in International Publication No. WO 2005 / 069916 (Appln. No. PCT / US2005 / 001581, WO 2008 / 116135 (Appln. No. PCT / US08 / 57786), W02010 / 132507 (Appln. No. PCT / US2010 / 034453), WO 2011 / 112900 (Appln. No. PCT / US2011 / 028042), and WO2012 / 174559 (Appln. No. PCT / US2012 / 043001) the entire contents of each of which are expressly incorporated by reference herein. Coenzyme Q10 is one of a series of polyprenyl 2,3-dimethoxy-5-methylbenzoquinone (ubiquinone) present in the mitochondrial electron transport systems of eukaryotic cells. Human cells produce coenzyme Q10 exclusively and it is found in cell and mitochondrial membranes of all human cells, with the highest levels in organs with high energy requirements, such as the liver and the heart. The body pool of coenzyme Q10 has been estimated to be about 2 grams, of which more than 50% is endogenous. Approximately 0.5 grams of coenzyme Q10 is required from the diet or biosynthesis each day.Coenzyme Q10 is produced in ton quantities from the worldwide supplement market and can be obtained from Kaneka, with plants in Pasadena, Texas and Takasagoshi, lapan.

[0319] The compositions containing a CoQlO compound may be used for the treatment and prevention of cancer. The compositions containing a CoQlO compound can be selfadministered by a patient, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s). In treating a patient exhibiting a disorder of interest, e.g., cancer, a therapeutically effective amount of the CoQlO compound is administered. A therapeutically effective dose refers to that amount of the compound which results in at least stable disease or a prolongation of survival in a patient.

[0320] Suitable routes of administration of the present compositions of the invention may include parenteral delivery, including, intravenous infusion, preferably continuous infusion, hi a preferred embodiment, the IV infusion comprises the active agent, e.g., coenzyme Q10, at approximately a 40 mg / mL (4% w / v) concentration. Where the composition is administered by IV infusion, it can be diluted in a pharmaceutically acceptable aqueous solution such as phosphate buffered saline or normal saline. In some embodiments, one or more routes of administration may be combined, such as, for example, intravenous and intratumoral, or intravenous and peroral, or intravenous and oral, or intravenous and topical, transdermal, or transmucosal. However, the methods provided herein include administration of coenzyme Q10 by continuous intravenous infusion.

[0321] For example, a CoQi 0 compound can be formulated for parenteral delivery, e.g., for intravenous injection. The compositions may be administered in a single bolus, multiple injections, or by continuous infusion (for example, intravenously or by peritoneal dialysis). For parenteral administration, the compositions may be formulated in a sterilized pyrogen-free form.

[0322] Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed, for the practice of the present invention, into dosages suitable for systemic administration is within the scope of the present disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.

[0323] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds which exhibit large therapeutic indices may be desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage form employed and the route of administration utilized.Formulations

[0324] The active agent, e.g., a CoQlO compound, can be delivered in any pharmaceutically acceptable carrier for the desired route of administration. As used herein, formulations including CoQlO compounds are formulated for administration by injection or infusion. In certain embodiments, the CoQlO compounds are not delivered orally.

[0325] Preferred therapeutic formulations for use in the methods of the invention comprise the active agent (e.g., a CoQlO compound) in a microparticle formation, e.g., for intravenous administration. Such intravenous formulations are provided, for example, in WO2011 / 112900 (Appln. No. PCT / US2011 / 028042), the entire contents of which arc expressly incorporated herein by reference, and an exemplary intravenous formulation of Coenzyme Q10 as described in WO2011 / 112900 (Appln. No. PCT / US2011 / 028042) is used in the examples set forth below. Through high pressure homogenization, active agent (e.g., a CoQlO compound) particles are reduced to produce particles that are small enough to pass through a 200-nm sterilizing filter. Particles that are small enough to pass through a 200-nm sterilizing filter can be injected intravenously. These particles are much smaller than blood cells and therefore will not embolize capillaries. Red blood cells for example are 6-micron x 2-micron disks. The particles are dispersed to and are encased or surrounded by a stabilizing agent. While not wishing to be bound by any theory, it is believed that the stabilizing agents are attracted to the hydrophobic therapeutic agent such that the dispersed particles of the hydrophobic therapeutic agent are surrounded by the stabilizing agent forming a suspension or an emulsion. The dispersed particles in the suspension or emulsion comprises a stabilizing agent surface and a core consisting of the hydrophobic therapeutic agent, e.g., a CoQlO compound, in a solid particulate form (suspension) or in an immiscible liquid form (emulsion). The dispersed particles can be entrenched in the lipophilic regions of a liposome.

[0326] Dispersed colloidal systems permit a high drug load in the formulation without the use of co-solvents. Additionally, high and relatively reproducible plasma levels areachieved without the dependence on endogenous low-density lipoprotein carriers. More importantly, the formulations allow sustained high drug levels in tumor cells due to the passive accumulation of the colloidal particles of the hydrophobic therapeutic agent.

[0327] A preferred intravenous formulation substantially comprises a continuous phase of water and dispersed solids (suspension) or dispersed immiscible liquid (emulsion). Dispersed colloidal systems, in which the particles are composed largely of the active agent (drug) itself, can often deliver more drug per unit volume than continuous solubilizing systems, if the system can be made adequately stable.

[0328] As the formulation medium, the aqueous solution may include Hank’s solution, Ringer’s solution, phosphate buffered saline (PBS), physiological saline buffer or other suitable salts or combinations to achieve the appropriate pH and osmolarity for parenterally delivered formulations. Aqueous solutions can be used to dilute the formulations for administration to the desired concentration. For example, aqueous solutions can be used to dilute a formulation for intravenous administration from a concentration of about 4% w / v to a lower concentration to facilitate administration of lower doses of coenzyme Q10. The aqueous solution may contain substances which increase the viscosity of the solution, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0329] The active agent (e.g., a CoQlO compound) is dispersed in the aqueous solution such that a colloidal dispersion is formed wherein the nano-dispersion particles of the hydrophobic therapeutic agent are covered or encased or encircled by the dispersion stabilizing agents to form nano-dispersions of the active agent e.g., a CoQlO compound) particles. The nano-dispersed active agent (e.g., a CoQlO compound) particles have a core formed of the hydrophobic therapeutic agent that is surrounded by the stabilizing agent. Similarly, in certain aspects, the stabilizing agent is a phospholipid having both a hydrophilic and lipophilic portion. The phospholipids form liposomes or other nanoparticles upon homogenization. In certain aspects these liposomes are bi-layered unilamellar liposomes while in other embodiments the liposomes are bi-layered multi- lamellar liposomes. The dispersed active agent (e.g., a CoQlO compound) particles are dispersed in the lipophilic portion of the bi-layered structure of the liposome formed from the phospholipids. In certain other aspects the core of the liposome, like the core of thenano-dispersion of active agent (e.g., a CoQlO compound) particles, is formed of the hydrophobic therapeutic agent and the outer layer is formed of the bi-layered structure of the phospholipid. In certain embodiments the colloidal dispersions are treated by a lyophilization process whereby the nanoparticle dispersion is converted to a dry powder.

[0330] In some embodiments, the formulation for injection or infusion used is a 4% sterile aqueous colloidal dispersion containing coenzyme Q10 in a nanosuspension as prepared in WO2011 / 112900. In certain embodiments, the formulation includes an aqueous solution; a hydrophobic active agent, e.g., coenzyme Q10, a coenzyme Q10 precursor or metabolite or a coenzyme Q10 related compound, dispersed to form a colloidal nano-dispersion of particles; and at least one of a dispersion stabilizing agent and an opsonization reducer; wherein the colloidal nano-dispersion of the active agent is dispersed into nano-dispersion particles having a mean size of less than 200 nm.

[0331] In certain embodiments, the dispersion stabilizing agent includes, but is not limited to, pegylated castor oil, Cremphor® EL, Cremophor® RH 40, Pegylated vitamin E, Vitamin E TPGS, and Dimyristoylphosphatidyl choline (DMPC).

[0332] In certain embodiments, the opsonization reducer is a poloxamer or a poloxamines.

[0333] In certain embodiments, the colloidal nano-dispersion is a suspension or an emulsion. Optionally, a colloidal nano-dispersion is in a crystalline form or a supercooled melt form.

[0334] In certain embodiments, the formulation for injection or infusion includes a lyoprotectant such as a nutritive sugar including, but not limited to, lactose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, glucosamine, galactosamine, N-methylglucosamine, mannitol, sorbitol, arginine, glycine, and sucrose; or any combination thereof.

[0335] In certain embodiments, the formulation for injection or infusion includes an aqueous solution; a hydrophobic active agent dispersed to form a colloidal nanodispersion of particles; and at least one of a dispersion stabilizing agent and an opsonization reducer. The colloidal nano-dispersion of the active agent is dispersed into nano-dispersion particles having sizes of less than 200 nm. In some embodiments the dispersion stabilizing agent is selected from natural or semisynthetic phospholipids. Forexample, suitable stabilizing agents include polyethoxylated (a / k / a pegylated) castor oil (Cremophor® EL), polyethoxylated hydrogenated castor oil (Cremophor® RH 40), Tocopherol polyethylene glycol succinate (Pegylated vitamin E, Vitamin E TPGS), Sorbitan fatty acid esters (Spans®), bile acids and bile-acid salts, or dimyristoylphosphatidyl choline (DMPC). In some embodiments the stabilizing agent is DMPC.

[0336] In certain embodiments the formulation is suitable for parenteral administration, including intravenous, intraperitoneal, orthotopical, intracranial, intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intranasal, or intraocular injections. In certain embodiments, the formulation contains coenzyme Q10, dimyristoyl-phophatidylcholine, and poloxamer 188 in a ratio of 4:3:1.5 respectively that is designed to stabilize the nanosuspension of the particles. In some embodiments, the formulation includes a phosphate buffer saline solution which contains sodium phosphate dibasic, potassium phosphate monobasic, potassium chloride, sodium chloride, and water for injection. In certain embodiments, the 4% sterile aqueous colloidal dispersion containing coenzyme Q10 in a nanosuspension is diluted in the phosphate buffered saline solution provided, e.g., 1:1, 1:2, 1:3, 1:4. 1:5, 1:6, 1:7, 1:8. 1:9, 1:10, 1:11, 1:12, 1:13, 1:14. 1:15, 1:16, 1:17, 1:18. 1:19, 1:20, or other appropriate ratio bracketed by any two of the values.

[0337] In some embodiments, a formulation for administration for use in the invention may include from about 0.001% to about 20% (w / w) of coenzyme Q10, about 0.01% to about 20% (w / w) of coenzyme Q10, about 0.1% to about 20% (w / w) of coenzyme Q10, more preferably about 0.01% to about 15% and even more preferably about 0.1% to about 10% (w / w) of coenzyme Q10. In certain embodiments, a formulation for any route of administration for use in the invention may include from about 1% to about 10% (w / w) of coenzyme Q10. In certain embodiments, a formulation for any route of administration for use in the invention may include from about 2% to about 8% (w / w) of coenzyme Q10. In certain embodiments, a formulation for any route of administration for use in the invention may include from about 2% to about 7% (w / w) of coenzyme Q10. In certain embodiments, a formulation for any route of administration for use in the invention may include from about 3% to about 6% (w / w) of coenzyme Q 10. Incertain embodiments, a formulation for any route of administration for use in the invention may include from about 3% to about 5% (w / w) of coenzyme Q10. In certain embodiments, a formulation for any route of administration for use in the invention may include from about 3.5% to about 4.5% (w / w) of coenzyme Q10. In certain embodiments, a formulation for any route of administration for use in the invention may include from about 3.5% to about 5% (w / w) of coenzyme Q10. In one embodiment a formulation includes about 4% (w / w) of coenzyme Q10. In one embodiment a formulation includes about 8% (w / w) of coenzyme Q10. In various embodiments, the formulation includes about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% (w / w) of coenzyme Q10, or any range bracketed by any two values recited. In certain embodiments, the formulations can be prepared as a percent weight to volume rather than a percent weight to weight.Depending on the formulation, the concentration of coenzyme Q10 may be the same, or about the same in the w / w and the w / v percent formulations. Coenzyme Q10 can be obtained from Kaneka Q10 as Kaneka Q10 (USP UBIDECARENONE) in powdered form (Pasadena, Texas, USA). Coenzyme Q10 used in the methods exemplified herein have the following characteristics: residual solvents meet USP 467 requirement; water content is less than 0.0%, less than 0.05% or less than 0.2%; residue on ignition is 0.0%, less than 0.05%, or less than 0.2% less than; heavy metal content is less than 0.002%, or less than 0.001%; purity of between 98-100% or 99.9%, or 99.5%.

[0338] In certain embodiments, the concentration of coenzyme Q10 in the formulation is 1 mg / mL to 150 mg / mL. In one embodiment, the concentration of coenzyme Q10 in the formulation is 5 mg / mL to 125 mg / mL. In one embodiment, the concentration of coenzyme Q10 in the formulation is 10 mg / mL to 100 mg / mL. In one embodiment, the concentration of coenzyme Q10 in the formulation is 20 mg / mL to 90 mg / mL. In one embodiment, the concentration of coenzyme Q10 is 30 mg / mL to 80 mg / mL. In one embodiment, the concentration of coenzyme Q10 is 30 mg / mL to 70 mg / mL. In one embodiment, the concentration of coenzyme Q10 is 30 mg / mL to 60 mg / mL. In one embodiment, the concentration of coenzyme Q10 is 30 mg / mL to 50 mg / mL. In one embodiment, the concentration of coenzyme QI 0 is 35 mg / mL to 45 mg / mL. It shouldbe understood that additional ranges having any one of the foregoing values as the upper or lower limits are also intended to be part of this invention, e.g., 10 mg / mL to 50 mg / mL, or 20 mg / mL to 60 mg / mL.

[0339] In certain embodiments, the concentration of coenzyme Q10 in the formulation is about 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 mg / mL. In one embodiment, the concentration of coenzyme Q10 in the formulation is about 50 mg / mL. In one embodiment, the concentration of coenzyme Q10 in the formulation is about 60 mg / mL. In one embodiment, the concentration of coenzyme Q10 in the formulation is about 30 mg / mL. In a preferred embodiment, the concentration of coenzyme Q10 in the formulation is about 40 mg / mL. It should be understood that ranges having any one of these values as the upper or lower limits are also intended to be part of this invention, e.g. between 37 mg / mL and 47 mg / mL, or between 31 mg / mL and 49 mg / mL.

[0340] It is understood that formulations can similarly be prepared containing coenzyme Q10 precursors, metabolites, and related compounds.H. KITS / PANELS

[0341] The invention also provides compositions and kits for prognosing or monitoring a disease or disorder, progression or recurrence of a disorder, response of a disease or disorder to a treatment, or survival of a subject being treated for a disorder e.g., pancreatic cancer). These kits may include one or more of the following: a reagent that specifically binds to a marker of the invention, and a set of instructions for measuring the level of the marker.

[0342] The invention also encompasses kits for detecting the presence of a marker in a biological sample, and / or prognosing a response to a Coenzyme Q10 treatment. Such kits can be used to determine if a subject is responsive to Coenzyme Q10 treatment. For example, the kit can comprise a labeled compound or agent capable of detecting a marker protein or nucleic acid in a biological sample and means for determining the amount of the protein or mRNA in the sample (e.g., an antibody which binds the protein or a fragment thereof, or an oligonucleotide probe which binds to DNA or mRNA encoding the protein). Kits can also include instructions for use of the kit for practicing any of themethods provided herein or interpreting the results obtained using the kit based on the teachings provided herein. The kits can also include reagents for detection of a control protein in the sample, e.g., actin for tissue samples, albumin in blood or blood derived samples for normalization of the amount of the marker present in the sample. The kit can also include the purified marker for detection for use as a control or for quantitation of the assay performed with the kit.

[0343] Kits include a panel of reagents for use in a method to prognose a response to a Coenzyme Q10 treatment for pancreatic cancer in a subject, the panel comprising one or more detection reagents, wherein each detection reagent is specific for the detection of a marker, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI- 18:0 / 22:4, PI- 18:0 / 20:2, PA-PI 8:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38.1.

[0344] For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a first marker protein; and, optionally, (2) a second, different antibody which binds to either the first marker protein or the first antibody and is conjugated to a detectable label. In certain embodiments, the kit includes (1) a second antibody (e.g., attached to a solid support) which binds to a second marker protein; and, optionally, (2) a second, different antibody which binds to either the second marker protein or the second antibody and is conjugated to a detectable label. The first and second marker proteins are different. In an embodiment, the first and second markers are markers of the invention, e.g., one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PL 18:0 / 22:4, PL 18:0 / 20:2, PA-P18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC- 38:8 / 0-38.1. In certain embodiments, the kit comprises a third antibody which binds to a third marker protein which is different from the first and second marker proteins, and a second different antibody that binds to either the third marker protein or the antibody that binds the third marker protein wherein the third marker protein is different from the first and second marker proteins.

[0345] For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleicacid sequence encoding a marker protein or (2) a pair of primers useful for amplifying a marker nucleic acid molecule. In certain embodiments, the kit can further include, for example: (1) an oligonucleotide, e.g., a second delectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a second marker protein or (2) a pair of primers useful for amplifying the second marker nucleic acid molecule. The first and second markers are different. In an embodiment, the first and second markers are markers of the invention, e.g., one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1. In certain embodiments, the kit can further include, for example: (1) an oligonucleotide, e.g., a third delectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a third marker protein or (2) a pair of primers useful for amplifying the third marker nucleic acid molecule wherein the third marker is different from the first and second markers. In certain embodiments, the kit includes a third primer specific for each nucleic acid marker to allow for detection using quantitative PCR methods.

[0346] For chromatography methods, the kit can include markers, including labeled markers, to permit detection and identification of one or more markers of the invention, e.g., one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1, by chromatography. In certain embodiments, kits for chromatography methods include compounds for derivatization of one or more markers of the invention. In certain embodiments, kits for chromatography methods include columns for resolving the markers of the method.

[0347] Reagents specific for detection of a marker of the invention, e.g., one or more markers of the invention, allow for detection and quantitation of the marker in a complex mixture, e.g., serum, tissue sample. In certain embodiments, the reagents are species specific. In certain embodiments, the reagents are not species specific. In certain embodiments, the reagents are isoform specific. In certain embodiments, the reagents are not isoform specific.

[0348] In certain embodiments, the kits comprise at least one reagent specific for the detection of the level of one or more markers of the invention. In certain embodiments,the kits further comprise instructions for the prognosis, monitoring, or characterization of a response to a Coenzyme Q10 treatment for pancreatic cancer based on the level of the at least one marker of the invention. In certain embodiments, the kits further comprise instructions to detect the level of a known marker in a sample in which the at least one marker of the invention is detected. In certain embodiments, the kits further comprise at least one reagent for the specific detection of a known marker.

[0349] The invention provides kits comprising at least one reagent specific for the detection of a level of at least one marker of the invention and at least one reagent specific for the detection of a level of a known marker.

[0350] In certain embodiments, the kits can also comprise, e.g., a buffering agents, a preservative, a protein stabilizing agent, reaction buffers. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kit can also contain a control sample or a scries of control samples which can be assayed and compared to the test sample. The controls can be control serum samples or control samples of purified proteins or nucleic acids, as appropriate, with known levels of target markers. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

[0351] The kits of the invention may optionally comprise additional components useful for performing the methods of the invention.

[0352] The invention further provides panels of reagents for detection of one or more markers of the invention in a subject sample and at least one control reagent. In certain embodiments, the marker comprises at least two or more markers, wherein each of the two or more markers are selected from the markers of the invention.

[0353] In certain embodiments, the control reagent is to detect the marker for detection in the biological sample wherein the panel is provided with a control sample containing the marker for use as a positive control and optionally to quantitate the amount of marker present in the biological sample. In certain embodiments, the panel includes a detection reagent for a maker known to be present or absent in the biological sample to provide a positive or negative control, respectively. The panel can be provided with reagents for detection of a control protein in the sample, e.g., actin for tissue samples, albumin inblood or blood derived samples for normalization of the amount of the marker present in the sample. The panel can be provided with a purified marker for detection for use as a control or for quantitation of the assay performed with the panel.

[0354] In certain embodiments, the level of the marker in the panel is increased when compared to a control or a predetermined threshold value. In certain embodiments, the level of the marker in the panel is decreased when compared to a control or a predetermined threshold value.

[0355] In some embodiments, the panel comprises one or more markers with an increased level when compared to a control or a predetermined threshold value, and / or one or more markers with a decreased level when compared to a control or a predetermined threshold value.

[0356] In a preferred embodiment, the panel includes reagents for detection of two or more markers of the invention (e.g., 2, 3, 4, 5, 6, 7, 8, 9), preferably in conjunction with a control reagent. In the panel, each marker is detected by a reagent specific for that marker. In certain embodiments, the panel further includes a reagent for the detection of a known marker. In certain embodiments, the panel includes replicate wells, spots, or portions to allow for analysis of various dilutions (e.g., serial dilutions) of biological samples and control samples. In a preferred embodiment, the panel allows for quantitative detection of one or more markers of the invention.

[0357] In certain embodiments, the panel is a protein chip for detection of one or more markers. In certain embodiments, the panel is an ELISA plate for detection of one or more markers. In certain embodiments, the panel is a plate for quantitative PCR for detection of one or more markers.

[0358] In certain embodiments, the panel of detection reagents is provided on a single device including a detection reagent for one or more markers of the invention and at least one control sample. In certain embodiments, the panel of detection reagents is provided on a single device including a detection reagent for two or more markers of the invention and at least one control sample. In certain embodiments, multiple panels for the detection of different markers of the invention are provided with at least one uniform control sample to facilitate comparison of results between panels.

[0359] The contents of all documents cited or referenced herein and all documents cited or referenced in the herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, GenBank Accession and Gene numbers, and published patents and patent applications, are hereby incorporated by reference, and may be employed in the practice of the invention. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.

[0360] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references and published patents and patent applications cited throughout the application arc hereby incorporated by reference.EXAMPLES

[0361] Example 1. Predictive Multi-omics Analysis of Coenzyme Q10 in Combination with Gemcitabine in a Phase 2 Study of Advanced Pancreatic Ductal Adenocarcinoma (PDAC).

[0362] Advanced PDAC has limited therapeutic options. Coenzyme Q10 was evaluated in Phase 2 in patients with advanced PDAC. In addition to response and safety, the study included FDG-PET and omics as exploratory analyses. Coenzyme Q10 was administered in an intravenous formulation as described in WO2011 / 112900 (Appln. No.PCT / US2011 / 028042) and US Patent No. 11 / 400,058, the entire contents of which are expressly incorporated herein. Multi-omics analyses from longitudinally collected patient samples including a subset of Adequately Treated Analysis Set (ATAS) (n=19) were performed to determine potential biomarkers associated with clinical outcomes.

[0363] Study Design and Methods: Metastatic PDAC patients (N=45) who had previously received between one and three lines of prior therapy were enrolled in this study. Omics analyses including proteomic, structural lipidomic, signaling lipidomic, and metabolomic measurements were performed on patient buffy coat and plasmasamples. Biomolecules with significant association to patient outcome (PFS, OS & TTP) were identified by linear regression analysis. Additional analysis with mixed effect linear modeling approach was used to relate omic values during ten weeks of treatment and patient ATAS status and time on study. Previous analysis of proteomics from patients in a phase 1 solid tumor study was mapped onto a similar omics output for phase 2 to reveal predictive biomarkers for disease status. In the phase 1 solid tumor study, patient samples were collected prior to start of Coenzyme Q10 treatment and subjected to multi-omics profiling. Following multi-omics profiling, differential expression analysis identified 406 molecular markers with predictive utility to patient response. These markers, where available, were evaluated in the phase 2 PDAC study for their utility to predict clinical outcome (best RECIST response during study, or RECIST status at cycle 2).

[0364] Results'. Proteomics analysis on patient huffy coats revealed enrichment of dicarboxylic and tricarboxylic metabolic processes, mitochondrial membrane space proteins, pyruvate metabolism, and malate / aspartate shuttle pathway with a positive correlation with TTP in the model, demonstrating an integrated connectivity to Coenzyme QlO’s functional effects on metabolism. Medium chain acyl carnitines and c- reactive protein (CRP) were identified as having the most significant differences in trajectories between patient ATC status by mixed effect linear modeling. Medium chain acyl carnitines differences could likely be the result of an integrated adaptation of liver homeostasis and physiological activity impacted by treatment.

[0365] Analysis of RECIST1.1 evaluated in Phase 1 clinical trial and subsequently validated in Phase 2 trial identified 4 proteins with predictive utility at the start of treatment (t=0) (SERPINA5, Vitronectin, Lumican, Proteosome 20S subunit alpha 4), 5 lipid molecular species (PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, SL-7-HDHA, and PC-38:8 / O-38:l), and 1 metabolite (2-keto-isovalerate).

[0366] Figure 1 shows the differential expression of protein, lipid, and metabolomic markers measured in the ATC population at baseline (prior to treatment with Coenzyme Q10). Differential expression analysis considered patient response to be either (A) “Best response during study,” which refers to the best RECIST status determined during the entire Coenzyme Q10 treatment, or (B) “Patient status at Cycle 2”, which refers to the RECIST status determined at Cycle 2 of Coenzyme QI 0 treatment. For each molecularmarker, the most significant clinical response is shown. Hence, SERPINA5, 2-keto- isovalerate, Proteosome 20S subunit alpha 4, SL-7-HDHA, PI-18:0 / 20:2, and PC- 38:8 / 0-38:1 are shown in terms of differential expression of “Best Response During Study,” while Vitronectin, Lumican, PI-18:0 / 22:4, and PA-P18:O / 18:O are shown in terms of differential expression of “Patient Status at Cycle 2.” Additionally, “Plasma,” and “Buffy Coat,” refer to the specific biological sample in which the marker was detected.The statistical significance of differential expression was assessed by Wilcoxon rank sum test. Phase 1 AUC refers to the results from differential expression analysis of the specified marker measured prior to start of Coenzyme Q10 treatment based on clinical response (length of time on Coenzyme Q10 treatment).

[0367] As shown in Figure 1, PDAC patients having a higher level of SERPINA5, Vitronectin, Lumican, 2-keto-isoval erate, PL18:0 / 22:4, PL18:0 / 20:2, or PA-PI 8:0 / 18:0 in plasma samples, or a lower level of Proteosome 20S subunit alpha 4, SL-7-HDHA, or PC-38:8 / O-38:l in plasma or buffy coat samples, were responsive to the Coenzyme Q10 treatment.

[0368] These markers exhibited differential expression in both clinical trials related to clinical outcome, thus demonstrating a biological relevance to Coenzyme Q10 treatment.

Claims

Claims1. A method for prognosing a response to Coenzyme Q10 treatment for pancreatic cancer in a subject, comprising:(a) detecting the level of one or more markers in a biological sample from the subject, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38:l; and(b) comparing the level of the one or more markers in the biological sample with a predetermined threshold value; wherein an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PL18:0 / 22:4, PI- 18:0 / 20:2, and / or PA-PI 8:0 / 18:0 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment; and / or wherein a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

2. The method of claim 1, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

3. The method of claim 1 or 2, wherein the Coenzyme Q10 treatment is administered by intravenous administration.

4. The method of any one of claims 1-3, wherein the Coenzyme Q10 treatment is administered by continuous infusion.

5. The method of claim 1, wherein an increase in the expression level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and / or PA- P18:O / 18:O relative to the predetermined threshold value, and / or a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to thepredetermined threshold value indicates that the subject will exhibit stable disease in response to the Coenzyme Q10 treatment.

6. The method of any one of claims 1-5, wherein a second drug is administered.

7. The method of claim 6, wherein the second drug is gemcitabine.

8. The method of any one of claims 1-7, wherein the response to the Coenzyme Q10 treatment comprises no change or a decrease in tumor size.

9. The method of any one of claims 1-8, wherein the response to the Coenzyme Q10 treatment comprises an increase in overall days of survival.

10. The method of any one of claims 1-9, wherein the biological sample comprises a blood sample or a component thereof.

11. The method of claim 10, wherein the sample comprises a buffy coat sample or a plasma sample.

12. The method of any one of claims 1-11, wherein the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers is determined.

13. The method of any one of claims 1-12, wherein the level of the one or more markers is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

14. The method of any one of claims 1-13, wherein the level of the one or more markers is detected by determining the level of its protein or corresponding mRNA in the biological sample.

15. The method of any one of claims 1-14, further comprising selecting and / or administering a treatment regimen based on the prediction of the response to Coenzyme Q10 treatment in the subject.

16. The method of claim 15, wherein the treatment regimen comprises further monitoring the subject for progression of pancreatic cancer.

17. The method of claim 15, wherein the treatment regimen is selected from the group consisting of (a) radiation therapy, (b) chemotherapy, (c) surgery, (d) hormone therapy, (e) antibody therapy, (f) immunotherapy, (g) cytokine therapy, (h) growth factor therapy, (i) watchful waiting, and (i) any combination of (a)-(i).

18. The method of any one of claims 1-17, wherein the subject has been previously diagnosed with pancreatic cancer.

19. A method for prognosing a response to Coenzyme Q10 treatment in a subject, wherein the prognosis is determined concurrently with the diagnosis of pancreatic cancer in the subject, comprising:(a) diagnosing the subject with pancreatic cancer;(b) detecting the level of one or more markers in a biological sample from the subject, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI- 18:0 / 20:2, PA- P18:0 / 18:0, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1; and(c) comparing the level of the one or more markers in the biological sample with a predetermined threshold value; wherein an increase in the level of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI- 18 :0 / 22:4, PI-18:0 / 20:2, and / or PA-P18:O / 18:O relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment; and / orwherein a decrease in the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 relative to the predetermined threshold value indicates that the subject will be responsive to the Coenzyme Q10 treatment.

20. The method of claim 19, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

21. The method of claim 19 or 20, wherein the Coenzyme Q10 treatment is administered by intravenous administration.

22. The method of any one of claims 19-21, wherein the Coenzyme Q10 treatment is administered by continuous infusion.

23. The method of claim 19, wherein a second drug is administered.

24. The method of claim 23, wherein the second drug is gemcitabine.

25. The method of any one of claims 19-24, wherein the response to the Coenzyme Q10 treatment comprises no change or a decrease in tumor size.

26. The method of any one of claims 19-25, wherein the response to the Coenzyme Q10 treatment comprises an increase in overall days of survival.

27. The method of any one of claims 19-26, wherein the biological sample comprises a blood sample or a component thereof.

28. The method of claim 27, wherein the sample comprises a buffy coat sample or plasma sample.

29. The method of any one of claims 19-28, wherein the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers, is determined.

30. The method of any one of claims 19-29, wherein the level of the one or more markers is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

31. The method of any one of claims 19-20, wherein the level of the one or more markers is detected by determining the level of its protein or corresponding mRNA in the biological sample.

32. The method of any one of claims 19-31 , further comprising selecting and / or administering a treatment regimen based on the prediction of the Coenzyme Q10 treatment in the subject.

33. The method of claim 32, wherein the treatment regimen comprises further monitoring of the subject for progression of cancer.

34. The method of claim 32, wherein the treatment regimen is selected from the group consisting of (a) radiation therapy, (b) chemotherapy, (c) surgery, (d) hormone therapy, (e) antibody therapy, (f) immunotherapy, (g) cytokine therapy, (h) growth factor therapy, (i) watchful waiting, and (i) any combination of (a)-(i).

35. The method of claim 17 or 34, wherein the treatment regimen comprises administering Coenzyme Q10, optionally in combination with gemcitabine.

36. A method for identifying an agent that modulates pancreatic cancer progression, comprising:(a) contacting a pancreatic cancer cell with a test compound,(b) determining the level of a marker in the cell, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PL18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1;(c) identifying an agent that modulates the level of the marker in the cell, thereby identifying an agent that modulates pancreatic cancer progression.

37. The method of claim 36, wherein the test compound is a small molecule, an antibody, or a nucleic acid inhibitor.

38. A compound identified by the method of claim 36.

39. A method of treating pancreatic cancer in a subject, comprising administering to the subject a modulator of a marker, wherein the marker is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-kcto-isovalcratc, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1.

40. The method of claim 39, wherein the modulator increases the marker level or activity.

41. The method of claim 39, wherein the modulator decreases the marker level or activity.

42. A kit for detecting a marker in a biological sample from a subject having pancreatic cancer, comprising one or more reagents for measuring the level of the marker in the biological sample from the subject, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PI- 18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1, and a set of instructions for measuring the level of the marker.

43. The kit of claim 42, wherein the reagent is an antibody that binds to the marker or an oligonucleotide that is complementary to the corresponding mRNA of the marker.Ill44. The kit of claim 42, wherein the instructions set forth an immunoassay, ELISA, or mass spectrometry assay for detecting the level of the marker in the biological sample.

45. The kit of claim 42, wherein the instructions set forth an amplification reaction for assaying the level of the mRNA in the biological sample corresponding to the marker.

46. The kit of claim 42, wherein the instructions set forth a hybridization assay for detecting the level of the mRNA in the biological sample corresponding to the marker.

47. The kit of claim 42, wherein the instructions further set forth comparing the level of the marker in the biological sample from the subject to a predetermined threshold value of the marker.

48. The kit of claim 42, wherein the marker comprises one or more markers with an increased level when compared to a predetermined threshold value, and / or one or more markers with a decreased level when compared to a predetermined threshold value.

49. A panel for use in a method of prognosing a response to a Coenzyme Q10 treatment for pancreatic cancer in a subject, the panel comprising one or more detection reagents, wherein each detection reagent is specific for the detection of a marker, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1.

50. A kit comprising the panel of claim 49 and a set of instructions for obtaining prognosis information based on a level of the marker.

51. A method for detecting a marker useful for prognosing a response to Coenzyme Q10 treatment for pancreatic cancer, comprising:(a) obtaining a sample from a subject diagnosed with pancreatic cancer; and(b) detecting the level of the marker in the biological sample, wherein the marker comprises one or more markers selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.1.

52. The method of claim 51, wherein the biological sample comprises a blood sample or a component thereof.

53. The method of claim 52, wherein the sample comprises a buffy coat sample or a plasma sample.

54. The method of any one of claims 51 -53, wherein the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers, is determined.

55. The method of any one of claims 51-54, wherein the level of the marker is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

56. The method of any one of claims 51-55, wherein the level of the marker is detected by determining the level of its protein or corresponding mRNA in the biological sample.

57. A method of treating pancreatic cancer in a subject comprising:(a) obtaining a biological sample from the subject;(b) submitting the biological sample to obtain prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto- isovalerate, PL18:0 / 22:4, PL18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2; and(c) administering a therapeutically effective amount of Coenzyme Q10 to the subject if the level of the one or more markers is above or below a predetermined threshold value.

58. A method of treating pancreatic cancer in a subject comprising:(a) obtaining prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PL18:0 / 22:4, PI-18:0 / 20:2, PA- P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2; and(b) administering a therapeutically effective amount of Coenzyme Q10 to the subject if the level of the one or more markers is above or below a predetermined threshold value.

59. A method of treating pancreatic cancer in a subject suspected of having pancreatic cancer, the method comprising:(a) obtaining a biological sample from the subject for use in identifying prognostic information as to the level of one or more markers in the biological sample, wherein the one or more markers is selected from the group consisting of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, PA-P18:O / 18:O, Proteosome 20S subunit alpha 4, SL-7-HDHA, and PC-38:8 / O-38.2;(b) measuring the level of the one or more markers in the biological sample; and(c) recommending to a healthcare provider to administer Coenzyme Q10 if the level of the one or more markers in the biological sample is above or below a predetermined threshold value.

60. The method of any one of claims 57-59, wherein the subject is prognosed to be responsive to the Coenzyme Q10 treatment if the level of SERPINA5, Vitronectin, Lumican, 2-keto-isovalerate, PI-18:0 / 22:4, PI-18:0 / 20:2, and / or PA-P18:O / 18:O is above the predetermined threshold value.

61. The method of any one of claims 57-59, wherein the subject is prognosed to be responsive to the Coenzyme Q10 treatment if the level of Proteosome 20S subunit alpha 4, SL-7-HDHA, and / or PC-38:8 / O-38.1 is below the predetermined threshold value.

62. The method of any one of claims 57-61, wherein the method further comprises administering a therapeutically effective amount of gemcitabine.

63. The method of any one of claims 57-62, wherein the biological sample comprises a blood sample or a component thereof.

64. The method of any one of claims 57-63, wherein the sample comprises a buffy coat sample or a plasma sample.

65. The method of any one of claims 57-64, wherein the level of at least two, three, four, five, six, seven, eight, nine or ten, of the markers, is determined.

66. The method of any one of claims 57-65, wherein the level of the one or more markers is detected by one or more of HPLC / UV-Vis spectroscopy, enzymatic analysis, mass spectrometry, NMR, immunoassay, ELISA, chromatography, or any combination thereof.

67. The method of any one of claims 57-66, wherein the level of the one or more markers is detected by determining the level of its protein or corresponding mRNA in the biological sample.