Ratio based biomarkers and methods of use thereof

Abstract

Compositions, methods and kits are described for identifying biomolecules (e.g., proteins and nucleic acids) expressed in a biological sample that are associated with the presence, development, or progression of a disease (such as cancer), or more generally determination of the etiology or risk factors associated with a disease. Sample types analyzed by the disclosed methods include but are not limited to archival tissue blocks that have been preserved in a fixative, tissue biopsy samples, tissue microarrays, and so forth. The methods disclosed herein correlate expression profiles of biomolecules with various disease types, and allow for the determination of relative survival rates; in some embodiments, the methods permit determination of survival rates for a subject with cancer. In other embodiments, the disclosure relates to methods for evaluating therapeutic regimes for the treatment, such as treatment of cancer.

1 .- 41 . (canceled) 
     
     
         42 . A method of determining survival or outcome probability for a subject with a disease, the method comprising:
 quantifying at least two disease associated proteins in a sample from the subject;   comparing the value of the first disease associated protein with the value of the second disease associated protein to obtain a biomarker indicator; and   correlating the biomarker indicator with survival or outcome probability of the subject with the disease when the biomarker reaches a predetermined cut-off value.   
     
     
         43 . The method of  claim 42 , further comprising:
 normalizing the at least two disease associated proteins in the sample to obtain a normalized value for each disease associated protein in the sample;   
       wherein the normalized value of the first disease associated protein is compared with the normalized value of the second disease associated protein to obtain the biomarker indicator. 
     
     
         44 . The method of  claim 43 , wherein the disease is a cancer and the method determines cancer diagnosis, prognosis, prediction of response, and/or relative survival rate for a subject with the cancer, the method comprising:
 quantifying the at least two disease associated proteins in the sample, wherein the disease associated proteins are cancer associated proteins;   normalizing the at least two cancer associated proteins in the sample to obtain a normalized value for each cancer associated protein in the sample;   comparing the normalized value of the first cancer associated protein with the normalized value of the second cancer associated protein to obtain a biomarker indicator; and   correlating the biomarker indicator with diagnosis, prognosis, prediction of response, and/or relative survival rate of the subject with cancer when the biomarker reaches a predetermined cut-off value.   
     
     
         45 . The method of  claim 44 , wherein the subject is a human or a non-human mammal. 
     
     
         46 . The method of  claim 44 , wherein the cancer is a solid tumor. 
     
     
         47 . The method of  claim 44 , wherein the cancer is selected from the group consisting of adrenal tumors, bile duct cancers, bladder cancer, bone cancer, brain tumors, breast cancer, cardiac sarcoma, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, germ cell cancers, gynecologic cancers, head and neck cancers, hepatoblastoma, renal cancer, laryngeal cancer, leukemias, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancers, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, skin cancer (non-melanoma), small bowel cancer, stomach (gastric) cancer, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, vulvar cancer, Wilms' tumor, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, liver carcinoma, stomach carcinoma, thyroid carcinoma, kidney carcinoma, bile duct carcinoma, and extrahepatic cholangiocarcinoma (EHCC). 
     
     
         48 . The method of  claim 44 , wherein at least one of the two cancer associated proteins comprises a tumor antigen selected from the group consisting of: 5T4; ACRBP; AKT; p-AKT; Blood Group Tn Antigen, CA150; CA19-9; CA50; CAB39L; CD20; CD22; CD24; CD63; CD66a+CD66c+CD66d+CD66e; CD164; CTAG1B; CTAG2; CTHRC1; Carcino Embryonic Antigen (CEA); EBAG9; EGFR; ErbB 2; FATE1; FLJ14868; FMNL1; GAGE1; GPA33; GPNMB; Galectin 8; Ganglioside OAcGD3; Heparanase 1; HER2; HER3; HORMAD1; JAKMIP2; LRIG3; Lung carcinoma Cluster 2; LYK5; M2A Oncofetal Antigen, MAGE 1; MAGEA10; MAGEA11; MAGEA12; MAGEA2; MAGEA4; MAGEA6; MAGEA8; MAGEA9; MAGEB1; MAGEB2; MAGEB3; MAGEB4; MAGEB6; MAGEB18; MAGEC1; MAGED2; MAGEE1; MAGEH1; MAGEL2; MelanA; Melanoma gp100; MGEA5; MOK protein kinase; MAPK; p-MAPK; mTOR; p-mTOR; MUC1; MUC2; MUC4; MUC16; Melanoma Associated Antigen; Mesothelin; Mucin 5AC; Neuroblastoma; NYS48; OCIAD1; OIP5; Ovarian Carcinoma-associated Antigen; PAGE4; PARP9; PATE; PCNA; PRAME; Plastin L; Prostate Mucin Antigen (PMA); Prostate Specific Antigen (PSA); Prostein; PTEN; RASD2; ROPN1; SART2; SART3; SBEM; SDCCAG10; SDCCAG8; SEPT1; SLC45A2; SPANX; SPANXB1; SSX5; STEAP4; STK31; TAG72 TBC1D2; TEM1; TRP1; XAGE2; Wilms' Tumor Protein, alpha 1 Fetoprotein; and tumor antigens of epithelial origin 
     
     
         49 . The method of  claim 44 , wherein at least one of the two cancer associated proteins is a AKT signaling pathway protein selected from the group consisting of: 4E-BP1, eIF-4E, AKT, Erk1/2, Hsp27, Hsp90, Tc11, Grb10, Ft1, Jip1, Posh, mTOR, periostin, and PTEN. 
     
     
         50 . The method of  claim 44 , wherein at least one of the two cancer associated proteins is a EGF signaling pathway protein selected from the group consisting of: EGF, EGFR, HER2, HER3, Beta-catenin, Glycoprotein 130, PLCG1, Erbin, MUC1, Grb2, Heat shock protein 90 kDa alpha (cytosolic), member A1, DLG4, PIK3R2, PICK1 and SHC1. 
     
     
         51 . The method of  claim 49 , wherein at least one of the two cancer associated proteins is a phosphorylated AKT signaling pathway protein selected from the group consisting of: phosphorylated 4e-BP1 (p-4E-BP1), phosphorylated eIF-4E (p-eIF-4E), phosphorylated AKT (pAKT), and phosphorylated mTOR (p-mTOR). 
     
     
         52 . The method of  claim 42 , wherein quantifying comprises analysis using immunohistochemistry. 
     
     
         53 . The method of  claim 44 , wherein quantifying comprises analysis using a stack of membranes, immunohistochemistry, laser capture micro dissection, “one dimensional” electrophoretic gel, a “two-dimensional” electrophoretic gel, mass-spectrometry, tissue microarray, protein microarray, multiplex tissue immunoblotting, or a combination of two or more thereof. 
     
     
         54 . The method of  claim 44 , wherein the biomarker indicator comprises a ratio of the normalized value of the first cancer associated protein divided by the normalized value of the second cancer associated protein. 
     
     
         55 . The method of  claim 44 , wherein the sample comprises a blood, sera, urine, or other fluid biological sample, or a tissue sample. 
     
     
         56 . The method of  claim 54 , wherein the tissue sample is selected from the group consisting of an archival tissue sample, a cryo-preserved tissue sample, a fresh tissue sample, an LCM tissue sample, or a tissue microarray. 
     
     
         57 . The method of  claim 44 , wherein the cancer comprises a solid tumor, and wherein the method comprises:
 obtaining a biomarker indicator using a method comprising:
 calculating the content of a first cancer associated protein in a solid tumor sample from the subject; 
 normalizing the first cancer associated protein content against total cellular protein content in the sample; 
 calculating the content of a second cancer associated protein in the solid tumor sample from the subject; 
 normalizing the second cancer associated protein content against total cellular protein content in the sample; and 
 correlating the normalized first cancer associated protein content against the normalized second cancer associated protein content to obtain the biomarker indicator; and 
   comparing the biomarker indicator with pre-determined prognosis or relative survival rates, thereby determining the prognosis or relative cancer survival rate for the subject with the solid tumor.   
     
     
         58 . A method for detecting cancer in a subject comprising:
 determining levels of a first cancer associated protein and a second cancer associated protein from a biological sample from the subject;   normalizing the first and second cancer associated protein content against total cellular protein content from the biological sample; and   comparing the normalized levels of the first and second cancer associated proteins with levels of the first and second cancer associated proteins in cells, tissues or bodily fluids measured in a control sample, wherein a decrease or increase in the normalized levels of the first and second cancer associated proteins in the subject versus levels of the first and second cancer associated proteins measured in the control sample beyond a predetermined cut-off value is associated with the presence of cancer in the subject.   
     
     
         59 . The method of  claim 58 , wherein at least one of the cancer associated proteins is p-AKT or p-mTOR, and the method comprises:
 normalizing the level of p-AKT or p-mTOR expression in the sample from the subject against total cellular protein content for the sample; and   comparing the level of normalized expression of p-AKT or p-mTOR in a control non-cancerous sample, wherein a statistically significant decrease in the level of p-AKT or p-mTOR expression in the subject's sample as compared to the control non-cancerous sample beyond a predetermined cut-off value is an indication of the presence of cancer in the subject.   
     
     
         60 . The method of  claim 58 , wherein the first and second cancer associated proteins comprise PTEN, p-AKT, p-mTOR, p-MAPK, EGFR, HER2, HERS, or a combination of two or more thereof. 
     
     
         61 . A survival-based cancer biomarker indicator, comprising at least two cancer associated proteins, wherein the at least two cancer associated proteins are two proteins from a cell signaling pathway associated with the cancer; and wherein the at least two cancer associated proteins are used to obtain the survival-based cancer biomarker indicator by:
 determining the level of the first cancer associated protein in a sample from a subject;   determining the level of the second cancer associated protein in the sample;   normalizing the first cancer associated protein level to total cellular protein content in the sample; and   normalizing the second cancer associated protein level to total cellular protein content in the sample, to obtain the survival-based cancer biomarker indicator.   
     
     
         61 . The survival-based cancer biomarker of  claim 61 , further comprising calculating a ratio of the normalized value of the first cancer associated protein divided by the normalized value of the second cancer associated protein to obtain the survival-based cancer biomarker indicator. 
     
     
         63 . A kit comprising:
 a membrane array for detecting cancer associated proteins in a sample, the array comprising a plurality of membranes, wherein each of the plurality of membranes has an affinity for one or more cancer associated proteins; and   containers of detector molecules for detecting the cancer associated proteins captured on at least one of the membranes;   
       wherein the cancer associated proteins are selected from the group of proteins associated with cancers consisting of solid tumors, leukemia, multiple myeloma or lymphoma. 
     
     
         64 . The kit of  claim 63 , wherein the cancer associated proteins are selected from the group of proteins associated with one or more of the following cancers: adrenal tumors, bile duct cancers, bladder cancer, bone cancer, brain tumors, breast cancer, cardiac sarcoma, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, germ cell cancers, gynecologic cancers, head and neck cancers, hepatoblastoma, renal cancer, laryngeal cancer, leukemia's, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancers, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, skin cancer (non-melanoma), small bowel cancer, stomach (gastric) cancer, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, vulvar cancer, Wilms' tumor, breast carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma, liver carcinoma, stomach carcinoma, thyroid carcinoma, kidney carcinoma, bile duct carcinoma, and extrahepatic cholangiocarcinoma (EHCC). 
     
     
         65 . The kit of  claim 63 , wherein the detector molecules comprise antibodies or probes specific for the detection of the cancer associated proteins, and the cancer associated proteins are selected from the group consisting of: 5T4; ACRBP; AKT; p-AKT; Blood Group Tn Antigen, CA150; CA19-9; CA50; CAB39L; CD20; CD22; CD24; CD63; CD66a+CD66c+CD66d+CD66e; CD164; CTAG1B; CTAG2; CTHRC1; Carcino Embryonic Antigen (CEA); EBAG9; EGFR; ErbB 2; FATE1; FLJ14868; FMNL1; GAGE1; GPA33; GPNMB; Galectin 8; Ganglioside OAcGD3; Heparanase 1; HER2; HER3; HORMAD1; JAKMIP2; LRIG3; Lung carcinoma Cluster 2; LYK5; M2A Oncofetal Antigen, MAGE 1; MAGEA10; MAGEA11; MAGEA12; MAGEA2; MAGEA4; MAGEA6; MAGEA8; MAGEA9; MAGEB1; MAGEB2; MAGEB3; MAGEB4; MAGEB6; MAGEB18; MAGEC1; MAGED2; MAGEE1; MAGEH1; MAGEL2; MelanA; Melanoma gp100; MGEA5; MOK protein kinase; MAPK; p-MAPK; mTOR; p-mTOR; MUC1; MUC2; MUC4; MUC16; Melanoma Associated Antigen; Mesothelin; Mucin 5AC; Neuroblastoma; NYS48; OCIAD1; 01P5; Ovarian Carcinoma-associated Antigen; PAGE4; PARP9; PATE; PCNA; PRAME; Plastin L; Prostate Mucin Antigen (PMA); Prostate Specific Antigen (PSA); Prostein; PTEN; RASD2; ROPN1; SART2; SART3; SBEM; SDCCAG10; SDCCAG8; SEPT1; SLC45A2; SPANX; SPANXB1; SSX5; STEAP4; STK31; TAG72 TBC1D2; TEM1; TRP1; XAGE2; Wilms' Tumor Protein, alpha 1 Fetoprotein; and tumor antigens of epithelial origin. 
     
     
         66 . The kit of  claim 63 , wherein the detector molecules comprise antibodies or probes for the detection of a cancer associated protein comprising at least one AKT signaling pathway protein selected from the group consisting of 4E-BP1, eIF-4E, AKT, Erk1/2, Hsp27, Hsp90, Tc11, Grb10, Ft1, Jip1, Posh, mTOR, periostin, and PTEN. 
     
     
         67 . The kit of  claim 63 , wherein detector molecules comprise antibodies or probes for the detection of a cancer associated protein comprising at least one phosphorylated AKT signaling pathway protein selected from the group consisting of phosphorylated 4E-BP1 (p-4E-BP1), phosphorylated eIF-4E (p-eIF-4E), phosphorylated AKT (pAKT), and phosphorylated mTOR (p-mTOR).