Method for biomarker and drug-target discovery for prostate cancer diagnosis and treatment as well as biomarker assays determined therewith

Abstract

The invention relates to a method for the determination of a cancer diagnostic/therapeutic biomarker assay and drug-targets including the following steps: (a) identification of potential candidate protein/peptide biomarkers and drug-targets based on the measurement of protein/peptide constituent concentrations in tissue sample proteomes as well as serum, plasma or any other derivatives of blood, or blood itself sample proteomes derived from healthy non-human mammalian individuals as well as from cancerous non-human mammalian individuals and qualitatively selecting as potential candidate protein/peptide biomarkers those which show a pronounced differential behaviour between healthy and cancerous sample proteomes; (b) optional verification of the potential candidate protein/peptide biomarkers as identified in step (a) by quantitative mass spectrometric measurement of the potential candidate protein biomarkers in serum, plasma or any other derivatives of blood, or blood itself sample proteomes derived from healthy non-human mammalian individuals as well as from cancerous non-human mammalian individuals and selecting as candidate protein/peptide biomarkers those which show a mass-spectrometrically measurable quantitative differential behaviour between healthy and cancerous sample proteomes; (c) validation of the candidate protein/peptide biomarkers as identified in step (a), or as optionally verified in step (b), by mass spectrometric measurement and/or antibody-based assays such as an Enzyme-Linked Immunosorbent Assay (ELISA) determination of the candidate protein biomarkers in serum, plasma or any other derivatives of blood, or blood itself sample proteomes derived from healthy human individuals as well as from cancerous human individuals and selecting as protein/peptide biomarkers those which show a mass-spectrometrically measurable and/or antibody-based assay detectable differential behaviour between healthy and cancerous sample proteomes; (d) application of statistical methods to uncover single or groups of protein/peptide biomarkers as validated in step (c) as signatures for the detection of patients with cancer. The invention furthermore relates to specific biomarker assays for the highly reliable diagnosis of cancer, specifically of localized or non-localized prostate cancer, using human serum, plasma or any other derivatives of blood, or blood itself.

1 - 16 . (canceled) 
     
     
         17 . A method for the determination of a cancer diagnostic or prognostic biomarker assay including the following steps:
 (a) identification of potential candidate protein or peptide biomarkers and drug-target based on the measurement of protein or peptide constituent concentrations in tissue sample proteomes as well as sample proteomes of serum, plasma or a derivative of blood, or blood itself derived from healthy non-human mammalian individuals as well as from cancerous non-human mammalian individuals and   qualitatively selecting as potential candidate protein/peptide biomarkers those which show a pronounced differential behaviour between healthy and cancerous sample proteomes;   (b) optional verification of the potential candidate protein/peptide biomarkers as identified in step (a) by quantitative mass spectrometric measurement of the potential candidate protein biomarkers in sample proteomes of serum, plasma or a derivative of blood, or blood itself derived from healthy non-human mammalian individuals as well as from cancerous non-human mammalian individuals and   selecting as candidate protein or peptide biomarkers those which show a mass-spectrometrically measurable quantitative differential behaviour between healthy and cancerous sample proteomes;   (c) validation of the candidate protein or peptide biomarkers as identified in step (a), or as optionally verified in step (b), by mass spectrometric measurement or affinity reagent-based determination of the candidate protein biomarkers in sample proteomes of serum, plasma or a derivative of blood, or blood itself derived from healthy human individuals as well as from cancerous human individuals and selecting as protein or peptide biomarkers those which show a mass-spectrometrically measurable or affinity reagent-based assay detectable differential behaviour between healthy and cancerous sample proteomes;   (d) application of statistical methods to uncover single or groups of protein or peptide biomarkers as validated in step (c) as signatures for the detection of patients with cancer or for the prognosis of cancer and/or the stratification of patients.   
     
     
         18 . The method according to  claim 17 , wherein the cancerous sample proteomes are sample proteomes of individuals with prostate cancer, and wherein the tissue samples are prostate tissue samples, and wherein the protein/peptide biomarkers are selected to be diagnostic of prostate cancer. 
     
     
         19 . The method according to  claim 17 , wherein the selection criteria for step (a) are selected from the group of:
 potential biomarkers regulated in prostate tissue and serum;   potential biomarkers regulated in prostate tissue and detected in serum;   potential biomarkers regulated in prostate tissue and secreted;   potential biomarkers exclusively detected in prostate tissue and sera of mice with cancer;   potential biomarkers, specific for prostate and regulated in cancer tissue or serum;   potential biomarkers specific for prostate and secreted;   potential biomarkers top regulated in prostate tissue or serum, by a factor of more than four;   potential biomarkers, prior knowledge-based selection, characterised by known biological function during cancer progression;   or a combination thereof.   
     
     
         20 . The method according to  claim 17 , wherein in step (a) the proteins or peptides of the digested proteins of the samples are in a first step identified by using a shotgun mass spectrometric technique, and in a second step a combined liquid chromatography/mass spectrometry technique, is used for the identification of the differential properties between healthy and cancerous samples. 
     
     
         21 . The method according to  claim 17 , wherein the affinity reagent-based method or assay is an antibody-based method or assay within step (c) and is selected to be an Enzyme-Linked Immunosorbent Assay (ELISA) or a Multiplex Bead Array Assay. 
     
     
         22 . A cancer diagnostic or therapeutic or prognostic or patient stratification biomarker assay determinable or determined using a method according to  claim 17 . 
     
     
         23 . A cancer diagnostic or therapeutic or prognostic or patient stratification biomarker assay for the diagnosis or therapy of prostate cancer comprising the measurement of at least two protein or peptide biomarkers determined according to a method according to  claim 17  in human serum, plasma or a derivative of blood, or blood itself. 
     
     
         24 . A method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer, for the distinction from benign prostate hyperplasia, using a combined measurement of the concentration of at least two, or at least three proteins or fragments of proteins selected from the group derived from: ASPN; VTN; AOC3; LOX; PGCP; PSAP; THBS1; CFH; CLU; KIT; TFRC; LGALS3BP; GOLPH2; HYOU1; CTSD; OLFM4; AKAP13; CP; CPE; CPM; ICAM1; MSMB; TM9SF3; GALNTL4 in human serum, plasma or a derivative of blood, or blood itself. 
     
     
         25 . The method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer according to  claim 24 , using a combined measurement of the concentration of ASPN derived protein or fragments thereof, in combination with VTN derived protein or fragments thereof, in human serum, plasma or a derivative of blood, or blood itself. 
     
     
         26 . A method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer for the distinction from benign prostate hyperplasia, using a combined measurement of the concentration of at least three proteins or fragments of proteins selected from the group derived from: ASPN; HYOU1; CTSD; OLFM4; in human serum, plasma or a derivative of blood, or blood itself, and wherein for the diagnosis/monitoring additionally the concentration of the Prostate Specific Antigen (PSA) in the human serum, plasma or a derivative of blood, or blood itself is measured using an affinity reagent-based assay. 
     
     
         27 . The method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer according to  claim 26 , wherein for a positive diagnosis or the monitoring of localized prostate cancer at least one or a combination of the following concentration criteria has to be met:
 the concentration of ASPN derived protein or fragments thereof has to be more than 55 ng/ml,   the concentration of HYOU1 derived protein or fragments thereof has to be more than 35 ng/ml,   the concentration of CTSD derived protein or fragments thereof has to be less than 32 ng/ml,   the concentration of OLFM4 derived protein or fragments thereof has to be less than 20 ng/ml.   
     
     
         28 . A method for the diagnosis or for the therapy or for the monitoring of non-localized prostate cancer, for the distinction from localized prostate cancer, using a combined measurement of the concentration of ASPN and CTSD and THBS1 and GALNTL4 as well as VTN derived protein or fragments thereof in human serum, plasma or a derivative of blood, or blood itself. 
     
     
         29 . The method for the diagnosis or for the therapy or for the monitoring of non-localized prostate cancer according to  claim 28 , for the distinction from localized prostate cancer using a combined measurement of the concentration of ASPN and CTSD and THBS1 and GALNTL4 as well as VTN derived protein or fragments thereof in human serum, plasma or a derivative of blood, or blood itself, in combination with the measurement of one further protein or fragments thereof selected from the group derived from: PSAP; GSPT1; CEACAM1; HYOU1; EFNA5; KIT. 
     
     
         30 . The method for the diagnosis and/or for the therapy or for the monitoring of non-localized prostate cancer according to  claim 28 , wherein for a positive diagnosis of metastatic prostate cancer the combination of the following concentration criteria has to be met:
 the concentration of ASPN derived protein or fragments thereof has to be more than 60 ng/ml and at the same time;   the concentration of CTSD derived protein or fragments has to be more than 120 ng/ml and at the same time;   the concentration of THBS1 derived protein or fragments has to be less than 12000 ng/ml and at the same time;   the concentration of GALNTL4 derived protein or fragments has to be more than 1400 ng/ml and at the same time; and   the concentration of VTN derived protein or fragments has to be more than 3000 ng/ml.   
     
     
         31 . The method according to  claim 24 , wherein for the diagnosis or monitoring additionally the concentration of the Prostate Specific Antigen (PSA) in the human serum, plasma or a derivative of blood, or blood itself is measured using an affinity reagent-based, namely an antibody-based Enzyme-Linked Immunosorbent Assay (ELISA), and wherein for a positive diagnosis the concentration of the Prostate Specific Antigen has to be more than 2 ng/ml, and wherein specifically for the diagnosis or monitoring of localized prostate cancer the concentration of the Prostate Specific Antigen has to be more than 2 ng/ml, and wherein for a positive diagnosis of non-localized prostate cancer the concentration of the prostate specific antigen has to be more than 100 ng/ml. 
     
     
         32 . The method according to  claim 24  for staging or diagnostic purposes, for the early detection and diagnosis of localized prostate cancer, or for therapy selection or for therapy monitoring. 
     
     
         33 . The method according to  claim 17 , wherein the cancerous sample proteomes are sample proteomes of individuals with prostate cancer, wherein the tissue samples are prostate tissue samples, wherein samples with localized or non-localized prostate cancer are used, and wherein the protein/peptide biomarkers are selected to be diagnostic of prostate cancer. 
     
     
         34 . The method according to  claim 17 , wherein in step (a) proteins derived from the sample proteomes are selected to be exclusively N-linked glycoproteins, and wherein in a first step the proteome of the sample is digested, and subsequently extracted using solid-phase extraction, and wherein the biomarkers are N-linked glycoproteins or peptide fragments thereof. 
     
     
         35 . The method according to  claim 17 , wherein the non-human mammalian individuals are mice, and wherein the mice prostate tissue for the samples in step (a) is perfused for complete removal of blood from the prostate tissue prior to the analysis or further treatment of the proteome. 
     
     
         36 . The method according to  claim 19 , wherein selection takes place if the factor between signals of healthy compared with signals of cancerous samples is larger than 1.5 or smaller than 0.75. 
     
     
         37 . The method according to  claim 19 , wherein selection takes place if the factor between signals of healthy compared with signals of cancerous samples is than 1.75 and smaller than 0.5. 
     
     
         38 . The method according to  claim 17 , wherein in step (b) absolute quantification is achieved by using a quantitative internal specifically synthesised internal standard. 
     
     
         39 . The method according to  claim 17 , wherein in step (b) or in step (c) selected reaction monitoring (SRM), optionally in combination with liquid chromatography, is used as mass spectrometry method. 
     
     
         40 . The method according to  claim 17 , wherein within step (a) the mass spectrometrically detected proteins or protein fragment signals are attributed to the corresponding proteins by using database information. 
     
     
         41 . A cancer diagnostic or therapeutic or prognostic or patient stratification biomarker assay for the diagnosis or therapy of prostate cancer comprising the measurement of at least three protein peptide biomarkers determined according to a method according  claim 17  in human serum, plasma or a derivative of blood, or blood itself, in combination with an affinity reagent-based antibody-based assay for the detection of Prostate Specific Antigen (PSA). 
     
     
         42 . The method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer, for the distinction from benign prostate hyperplasia, according to  claim 24  using a combined measurement of the concentration of or at least three proteins or fragments of proteins selected from the group derived from: ASPN; VTN; AOC3; LOX; PGCP; PSAP; THBS1; CFH; CLU; KIT; TFRC; LGALS3BP; GOLPH2; HYOU1; CTSD; OLFM4; AKAP13; CP; CPE; CPM; ICAM1; MSMB; TM9SF3; GALNTL4 in human serum, plasma or a derivative of blood, or blood itself. 
     
     
         43 . The method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer, for the distinction from benign prostate hyperplasia, according to  claim 24  the measurement is carried out using tandem mass spectrometry techniques, namely selected reaction monitoring (SRM), optionally in combination with liquid chromatography, aor Enzyme-Linked Immunosorbent Assays (ELISA) for the detection of these proteins or fragments thereof. 
     
     
         44 . The method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer according to  claim 24 , using a combined measurement of the concentration of ASPN derived protein or fragments thereof, optionally in combination with VTN derived protein or fragments thereof, in human serum, plasma or a derivative of blood, or blood itself, in combination with the measurement of one further protein or fragments thereof selected from the group derived from: AOC3; LOX; PGCP; PSAP; THBS1; CFH; CLU; KIT; TFRC; LGALS3BP; GOLPH2; HYOU1; CTSD; OLFM4. 
     
     
         45 . The method ethod for the diagnosis or for the therapy or for the monitoring of localized prostate cancer according to  claim 25 , wherein for a positive diagnosis or the monitoring of localized prostate cancer
 the concentration of ASPN derived protein or fragments thereof has to be more than 55 ng/ml, and at the same time, at least one of the following concentration criteria has to be met:   the concentration of VTN derived protein or fragments has to be less than 3500 ng/ml,   the concentration of AOC3 derived protein or fragments thereof has to be less than 250 ng/ml,   the concentration of LOX derived protein or fragments thereof has to be less than 580 ng/ml,   the concentration of PGCP derived protein or fragments thereof has to be more than 550 ng/ml,   the concentration of PSAP derived protein or fragments thereof has to be less than 33000 ng/ml,   the concentration of THBS1 derived protein or fragments thereof has to be more than 12500 ng/ml,   the concentration of LGALS3BP derived protein or fragments thereof has to be more than 390 ng/ml,   the concentration of GOLPH2 derived protein or fragments thereof has to be more than 80 ng/ml,   the concentration of HYOU1 derived protein or fragments thereof has to be more than 35 ng/ml,   the concentration of CTSD derived protein or fragments thereof has to be less than 32 ng/ml,   the concentration of OLFM4 derived protein or fragments thereof has to be less than 20 ng/ml.   
     
     
         46 . A method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer for the distinction from benign prostate hyperplasia, using a combined measurement of the concentration of at least three proteins or fragments of proteins selected from the group derived from: ASPN; HYOU1; CTSD; OLFM4; in human serum, plasma or a derivative of blood, or blood itself, and wherein for the diagnosis or monitoring additionally the concentration of the Prostate Specific Antigen (PSA) in the human serum, plasma or a derivative of blood, or blood itself is measured using an affinity reagent-based, an antibody-based assay, and wherein for a positive diagnosis the concentration of the Prostate Specific Antigen has to be more than 4 ng/ml. 
     
     
         47 . The method for the diagnosis or for the therapy or for the monitoring of localized prostate cancer according to  claim 26 , wherein for a positive diagnosis or the monitoring of localized prostate cancer at least one or a combination of the following concentration criteria has to be met:
 the concentration of ASPN derived protein or fragments thereof has to be more than 60 ng/ml,   and/or the concentration of HYOU1 derived protein or fragments thereof has to be more than 40 ng/ml,   and/or the concentration of CTSD derived protein or fragments thereof has to be less than 25 ng/ml,   and/or the concentration of OLFM4 derived protein or fragments thereof has to be less than 15 ng/ml.   
     
     
         48 . The method for the diagnosis or for the therapy or for the monitoring of non-localized prostate cancer, according to  claim 28 , wherein the measurement is carried out using tandem mass spectrometry techniques, namely selected reaction monitoring (SRM), optionally in combination with liquid chromatography, or Enzyme-Linked Immunosorbent Assays (ELISA) for the detection of these proteins or fragments thereof 
     
     
         49 . The method for the diagnosis or for the therapy or for the monitoring of non-localized prostate cancer according to  claim 28 , wherein for a positive diagnosis of metastatic prostate cancer at least one or a combination of the following concentration criteria has to be met:
 the concentration of ASPN derived protein or fragments thereof has to be more than 68 ng/ml and at the same time,   the concentration of CTSD derived protein or fragments has to be more than 133 ng/ml and at the same time,   the concentration of THBS1 derived protein or fragments has to be less than 10750 ng/ml and at the same time,   the concentration of GALNTL4 derived protein or fragments has to be more than 1650 ng/ml and at the same time,   the concentration of VTN derived protein or fragments has to be more than 3300 ng/ml,   wherein if, additionally measured,   the concentration of PSAP derived protein or fragments thereof has to be more than 34000 ng/ml,   or the concentration of GSPT1 derived protein or fragments thereof has to be more than 510 ng/ml,   or the concentration of CEACAM1 derived protein or fragments thereof has to be more than 38 ng/ml,   or the concentration of HYOU1 derived protein or fragments thereof has to be more than 89 ng/ml,   or the concentration of EFNA5 derived protein or fragments thereof has to be more than 65 ng/ml,   or the concentration of KIT derived protein or fragments thereof has to be more than 95 ng/ml.   
     
     
         50 . The method according to  claim 24 , wherein for the diagnosis or monitoring additionally the concentration of the Prostate Specific Antigen (PSA) in the human serum, plasma or a derivative of blood, or blood itself is measured using an affinity reagent-based, namely an antibody-based Enzyme-Linked Immunosorbent Assay (ELISA), and wherein for a positive diagnosis the concentration of the Prostate Specific Antigen has to be more than 4 ng/ml, and wherein specifically for the diagnosis/monitoring of localized prostate cancer the concentration of the Prostate Specific Antigen has to be more than more than 8 ng/ml, and wherein for a positive diagnosis of non-localized prostate cancer the concentration of the prostate specific antigen has to be more than 175 ng/ml. 
     
     
         51 . The method for the diagnosis and/or for the therapy or for the monitoring of non-localized prostate cancer according to  claim 29 , wherein for a positive diagnosis of metastatic prostate cancer the combination of the following concentration criteria has to be met:
 the concentration of ASPN derived protein or fragments thereof has to be more than 60 ng/ml and at the same time,   the concentration of CTSD derived protein or fragments has to be more than 120 ng/ml and at the same time,   the concentration of THBS1 derived protein or fragments has to be less than 12000 ng/ml and at the same time,   the concentration of GALNTL4 derived protein or fragments has to be more than 1400 ng/ml and at the same time,   the concentration of VTN derived protein or fragments has to be more than 3000 ng/ml.