Methods, arrays and uses thereof

Abstract

The present invention provides a method for diagnosing or determining a pancreatic cancer-associated disease state comprising or consisting of the steps of: (a) providing a sample from an individual to be tested; and (b) determining a biomarker signature of the test sample by measuring the presence and/or amount in the test sample of two or more biomarkers selected from the group defined in Table A; wherein the presence and/or amount in the test sample of two or more biomarkers selected from the group defined in Table A is indicative of the pancreatic cancer-associated disease state in the individual; uses and methods of determining a pancreatic cancer-associated disease state, and methods of treating pancreatic cancer, together with arrays and kits for use in the same.

1 . A method for diagnosing or determining a pancreatic cancer-associated disease state comprising or consisting of the steps of:
 (a) providing a sample from an individual to be tested; and   (b) measuring the presence and/or amount in the test sample of two or more biomarkers selected from the group defined in Table A;   
       wherein the presence and/or amount in the test sample of the two or more biomarkers selected from the group defined in Table A is indicative of the pancreatic cancer-associated disease state in the individual. 
     
     
         2 . The method according to  claim 1  wherein the pancreatic cancer-associated disease state is selected from the group consisting of:
 (i) diagnosis of pancreatic cancer; 
 (ii) diagnosis of early pancreatic cancer 
 
     
     
         3 . The method according to any one of the preceding claims wherein the pancreatic cancer-associated disease state is early pancreatic cancer. 
     
     
         4 . The method according to  claim 3  wherein the method is for the diagnosis of stage I or stage II pancreatic cancer. 
     
     
         5 . The method according to any previous claim wherein the method is for distinguishing an individual with a pancreatic cancer-associated disease state from an individual without pancreatic cancer but with symptoms suggestive or consistent with pancreatic cancer, optionally wherein the individual without pancreatic cancer has a benign pancreatic or biliary disease, e.g. acute and chronic pancreatitis, diabetes, liver disease, pancreatic cyst, gallstone disease and IgG4 disease. 
     
     
         6 . The method according to any previous claim wherein the sample in step (a) is from an individual in one or more of the following risk groups:
 (a) Individuals with a family history of pancreatic cancer or certain hereditary predispositions (e.g. Peutz-Jeghers syndrome);   (b) Individuals diagnosed with diabetes (e.g. new-onset diabetes, in particular those aged 50 years or over); and/or   (c) Individuals with symptoms suggestive or consistent with pancreatic cancer.   
     
     
         7 . The method according to any previous claim wherein the sample in step (a) is from an individual with a benign pancreatic or biliary disease, e.g. acute and chronic pancreatitis, diabetes, liver disease, pancreatic cyst, gallstone disease and IgG4 disease. 
     
     
         8 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of two or more biomarkers listed in Table A(i)-(vi), for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the biomarkers listed in Table A(i)-(vi). 
     
     
         9 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of six or more biomarkers listed in Table A, for example at least 7, 8, 9, 10, 11, 12 or 13 of the biomarkers listed in Table A. 
     
     
         10 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of three or more biomarkers listed in Table A(i)-(v), for example at least 4, 5, 6, 7, 8, or 9 of the biomarkers listed in Table A(i)-(v). 
     
     
         11 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of one or more biomarker(s) listed in Table A, part (i) and/or part (iii) and/or part (v). 
     
     
         12 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; and (ii) GSN and/or HADH2. 
     
     
         13 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) GSN and/or HADH2; and (iii) IGFBP3. 
     
     
         14 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) IGFBP3; and (iii) MUC16 and/or FCN2 and/or MASP2. 
     
     
         15 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) GSN and/or HADH2; (iii) IGFBP3, and (iv) Complement Factor B. 
     
     
         16 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of VWF, FCN2, and/or MASP2 
     
     
         17 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of two or more of the following biomarkers: (i) OPG and/or VWF; (ii) GSN and/or HADH2; (iii) IGFBP3; (iv) MUC16, FCN2, and/or MASP2. 
     
     
         18 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) GSN and/or HADH2; (iii) IGFBP3; (iv) Complement Factor B; (v) MUC16 and/or FCN2 and/or MASP2; (vi) Complement C4; (vii) Complement C5; and (viii) Cystatin C. 
     
     
         19 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of two or more of the following biomarkers and/or one or more secondary target(s) thereof: OPG, GSN, IGFBP3, Complement Factor B, MUC16, Complement C4, Complement C5, Cystatin C; optionally wherein the one or more secondary targets are selected from VWF, HADH2, FCN2, MASP2. 
     
     
         20 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of the following biomarkers: OPG, GSN, IGFBP3, Complement Factor B, MUC16, Complement C4, Complement C5, Cystatin C; and optionally including measuring the presence and/or amount of one or more biomarkers selected from VWF, HADH2, FCN2, MASP2. 
     
     
         21 . The method according to any one of the preceding claims wherein step (b) comprises measuring the presence and/or amount of CA 19-9. 
     
     
         22 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) GSN and/or HADH2; and (iii) CA 19-9. 
     
     
         23 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) GSN and/or HADH2; (iii) IGFBP3; and (iv) CA 19-9. 
     
     
         24 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) IGFBP3; (iii) MUC16, FCN2 and/or MASP2; and (iv) CA 19-9. 
     
     
         25 . The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the presence and/or amount of: (i) OPG and/or VWF; (ii) GSN and/or HADH2; (iii) IGFBP3; (iv) Complement Factor B; (v) MUC16, FCN2 and/or MASP2; (vi) Complement C4; (vii) Complement C5; (viii) Cystatin C; and (ix) CA 19-9. 
     
     
         26 . The method according to any one of the preceding claims wherein step (b) comprises measuring the presence and/or amount of one or more biomarker(s) listed in Table 1, for example 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, or 33 of the biomarkers in Table 1. 
     
     
         27 . The method according to any one of the preceding claims wherein step (b) comprises measuring the presence and/or amount of all of the biomarkers listed in Table A (e.g. at the protein, mRNA and/or ctDNA level). 
     
     
         28 . The method according to any one of the preceding claims further comprising or consisting of the steps of:
 (c) providing one or more control samples from:
 i. an individual not afflicted with pancreatic cancer; and/or 
 ii. an individual afflicted with a benign pancreatic and/or biliary disease; and 
   (d) determining a biomarker signature of the one or more control samples by measuring the presence and/or amount in the control sample of the one or more biomarkers measured in step (b);   
       wherein the pancreatic cancer-associated disease state is identified in the event that the presence and/or amount in the test sample of the one or more biomarkers measured in step (b) is different from the presence and/or amount in the control sample of the one or more biomarkers measured in step (d). 
     
     
         29 . The method according to any one of the preceding claims further comprising or consisting of the steps of:
 (e) providing one or more control samples from an individual afflicted with pancreatic cancer; and   (f) determining a biomarker signature of the control sample by measuring the presence and/or amount in the control sample of the one or more biomarkers measured in step (b);   
       wherein the pancreatic cancer-associated disease state is identified in the event that the presence and/or amount in the test sample of the one or more biomarkers measured in step (b) corresponds to the presence and/or amount in the control sample of the one or more biomarkers measured in step (f). 
     
     
         30 . The method according to  claim 29  wherein the individual not afflicted with pancreatic cancer is a healthy individual. 
     
     
         31 . The method according to  claim 29  or  30  wherein the one or more individual afflicted with pancreatic cancer is afflicted with a pancreatic cancer selected from the group consisting of adenocarcinoma (e.g., pancreatic ductal adenocarcinoma or tubular papillary pancreatic adenocarcinoma), pancreatic sarcoma, malignant serous cystadenoma, adenosquamous carcinoma, signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, undifferentiated carcinoma, and undifferentiated carcinomas with osteoclast-like giant cells. 
     
     
         32 . The method according to any one of the preceding claims wherein the pancreatic cancer is pancreatic ductal adenocarcinoma. 
     
     
         33 . The method according to any one of the preceding claims wherein step (b) comprises measuring the expression of the protein or polypeptide of one or more biomarker(s). 
     
     
         34 . The method according to  claim 33  wherein step (b), (d) and/or step (f) is performed using one or more first binding agent capable of binding to a biomarker protein or polypeptide listed in Table A. 
     
     
         35 . The method according to  claim 34  wherein the one or more first binding agent comprises one or more of the antibody sequences defined in Table 5 and/or Table 6. 
     
     
         36 . The method according to  claim 35  wherein the one or more first binding agent comprises one or more antibody sequences selected from the group consisting of: SEQ ID NO: 6, 11, 13, 15, 20, 30, 32, and 36. 
     
     
         37 . The method according to  claim 35  wherein the one or more first binding agent comprises one or more of the antibody sequences SEQ ID NO: 30, 32, and 36. 
     
     
         38 . The method according to any one of  claims 33 - 37  wherein the first binding agent comprises or consists of an antibody or an antigen-binding fragment thereof. 
     
     
         39 . The method according to  claim 38  wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof. 
     
     
         40 . The method according to any one of  claims 35  to  38  wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule. 
     
     
         41 . The method according to any one of  claims 34  to  40  wherein the first binding agent is immobilised on a surface. 
     
     
         42 . The method according to one of the preceding claims wherein the one or more biomarkers in the test and/or control sample(s) are labelled with a detectable moiety. 
     
     
         43 . The method according to  claim 42  wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety. 
     
     
         44 . The method according to  claim 42  or  43  wherein the detectable moiety is biotin. 
     
     
         45 . The method according to any one of  claims 33  to  44  wherein step (b), (d) and/or step (f) is performed using an assay comprising a second binding agent capable of binding to the one or more biomarkers, the second binding agent comprising a detectable moiety. 
     
     
         46 . The method according to  claim 45  wherein the second binding agent comprises or consists of an antibody or an antigen-binding fragment thereof. 
     
     
         47 . The method according to  claim 46  wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof. 
     
     
         48 . The method according to  claim 46  or  47  wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: scFv; Fab; a binding domain of an immunoglobulin molecule. 
     
     
         49 . The method according to any one of  claims 45  to  48  wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety. 
     
     
         50 . The method according to  claim 49  wherein the detectable moiety is fluorescent moiety (for example an Alexa Fluor dye, e.g. Alexa647). 
     
     
         51 . The method according to any one of the preceding claims wherein the method comprises or consists of an ELISA (Enzyme Linked Immunosorbent Assay). 
     
     
         52 . The method according to any one of the preceding claims wherein step (b), (d) and/or step (f) is performed using an array. 
     
     
         53 . The method according to  claim 52  wherein the array is selected from the group consisting of: macroarray; microarray; nanoarray. 
     
     
         54 . The method according to any one of the preceding claims wherein the method comprises:
 (i) labelling biomarkers present in the sample with biotin;   (ii) contacting the biotin-labelled proteins with an array comprising a plurality of scFv immobilised at discrete locations on its surface, the scFv having specificity for one or more of the proteins in Table A(i)-(vi);   (iii) contacting the biotin-labelled proteins (immobilised on the scFv) with a streptavidin conjugate comprising a fluorescent dye; and   (iv) detecting the presence of the dye at discrete locations on the array surface   
       wherein the expression of the dye on the array surface is indicative of the expression of a biomarker from Table A in the sample. 
     
     
         55 . The method according to any one of  claims 1  to  32  wherein step (b), (d) and/or (f) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarkers. 
     
     
         56 . The method according to  claim 55 , wherein measuring the expression of the one or more biomarker(s) in step (b), (d) and/or (f) is performed using one or more binding agent, each individually capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table A. 
     
     
         57 . The method according to any one of the preceding claims wherein the sample provided in step (a), (c) and/or (e) is selected from the group consisting of unfractionated blood, plasma, serum, tissue fluid, pancreatic tissue, milk, bile and urine. 
     
     
         58 . The method according to  claim 57 , wherein the sample provided in step (a), (c) and/or (e) is selected from the group consisting of unfractionated blood, plasma and serum. 
     
     
         59 . The method according to  claim 57  or  58  wherein the sample provided in step (a), (c) and/or (e) is serum. 
     
     
         60 . The method according to any one of the preceding claims wherein the predictive accuracy of the method, as determined by an ROC AUC value, is at least 0.50, for example at least 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or at least 0.99. 
     
     
         61 . The method according to  claim 60  wherein the predictive accuracy of the method, as determined by an ROC AUC value, is at least 0.83. 
     
     
         62 . The method according to any one of the preceding claims further comprising one or more further clinical investigations (such as testing a biopsy sample and/or in vivo imaging of the patient) in order to confirm or establish the diagnosis. 
     
     
         63 . The method according to any one of the preceding claims wherein, in the event that the individual is diagnosed with pancreatic cancer, the method comprises step (g) of providing the individual with a pancreatic cancer therapy. 
     
     
         64 . The method according to  claim 63  wherein the pancreatic cancer therapy is selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, chemoimmunotherapy, thermochemotherapy and combinations thereof. 
     
     
         65 . The method according to  claim 63  or  64  wherein the pancreatic cancer therapy comprises or consists of surgical removal of the pancreas in whole or in part (for example, using the Whipple procedure to remove the pancreas head or a total pancreatectomy) combined with chemotherapy (for example, gemcitabine and/or 5-fluorouracil). 
     
     
         66 . The method according to any previous claim wherein measuring the presence and/or amount in the test sample of one or more biomarkers selected from the group defined in Table A in step (b) is replaced with measuring the presence and/or amount in the test sample of one or more protein bound by one or more of the antibody sequences described in Table 5. 
     
     
         67 . An array for determining the presence of, or risk of having, pancreatic cancer in an individual comprising an agent or agents for detecting the presence in a protein and/or nucleic acid sample from the individual of one or more of the biomarkers defined in Table A. 
     
     
         68 . The array according to  claim 67  wherein the agent or agents for detecting the presence in a sample of one or more of the biomarkers defined in Table A is/are one or more binding agents as defined in any one of  claim 34  to  44  or  56 . 
     
     
         69 . The array according to  claim 67  or  68  wherein the array comprises agents capable of binding to all of the biomarkers defined in Table A(i)-(vi); optionally wherein the array comprises agents capable of binding to all of the biomarkers defined in Table A. 
     
     
         70 . The array according to  claim 67  or  68  wherein the array comprises agents capable of binding to the following biomarkers:
 (i) OPG and/or VWF; (ii) GSN and/or HADH2; (iii) IGFBP3; (iv) Complement Factor B; (v) MUC16, FCN2 and/or MASP2; (vi) Complement C4; (vii) Complement C5; and (viii) Cystatin C; optionally including one or more additional biomarkers from Table 1. 
 
     
     
         71 . The array according to any one of  claims 67  to  70  wherein the array comprises antibodies, or antigen-binding fragments thereof, capable of binding to all of the biomarkers at the protein level. 
     
     
         72 . The array according to any one of  claims 67  to  71  wherein the array comprises one or more of the antibodies identified in Table 5. 
     
     
         73 . The array according any one of  claims 67  to  72  wherein the array comprises one or more of the antibodies in Table 6. 
     
     
         74 . Use of two or more biomarkers selected from the group defined in Table A as biomarkers for determining the presence of, or risk of having, pancreatic cancer in an individual. 
     
     
         75 . The use according to  claim 74  wherein the two or more biomarkers comprise the following biomarkers:
 (i) OPG and/or VWF; (ii) GSN and/or HADH2; (iii) IGFBP3; (iv) Complement Factor B; (v) MUC16, FCN2 and/or MASP2; (vi) Complement C4; (vii) Complement C5; and (viii) Cystatin C; optionally the use additionally including use of CA 19-9 and/or one or more additional biomarkers from Table 1. 
 
     
     
         76 . The use according to  claim 74  or  75  wherein all of the biomarkers defined in Table A are used together as a diagnostic signature for determining the presence of pancreatic cancer in an individual. 
     
     
         77 . A kit for determining the presence of, or risk of having, pancreatic cancer comprising:
 (a) an array according to any one of  claims 67  to  73 , or components for making the same; and   (b) instructions for performing the method as defined in any one of  claims 1  to  66 .   
     
     
         78 . A method of treating pancreatic cancer in an individual comprising the steps of:
 (a) diagnosing pancreatic cancer according to the method defined in any one of  claims 1  to  66 ; and   (b) providing the individual with pancreatic cancer therapy.   
     
     
         79 . The method according to  claim 78  wherein step (a) further comprises comprise one or more further clinical investigations (such as testing a biopsy sample and/or in vivo imaging of the patient) in order to confirm or establish the diagnosis. 
     
     
         80 . The method according to  claim 78  or  79  wherein the pancreatic cancer therapy is selected from the group consisting of surgery (e.g., resection), chemotherapy, immunotherapy, chemoimmunotherapy and thermochemotherapy. 
     
     
         81 . The method of any one of  claims 78  to  80  wherein the pancreatic cancer therapy comprises surgical removal of the pancreas in whole or in part (e.g. using the Whipple procedure to remove the pancreas head or a total pancreatectomy) combined with chemotherapy (e.g. gemcitabine and/or 5-fluorouracil). 
     
     
         82 . A method or use for determining the presence of pancreatic cancer in an individual substantially as described herein. 
     
     
         83 . An array or kit for determining the presence of pancreatic cancer in an individual substantially as described herein.