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 . A method of determining cancer prognosis, for a subject with gastric cancer, the method comprising:
 quantifying at least a first cancer associated protein and a second cancer associated protein in a gastric cell sample from the subject, wherein the first and second cancer associated proteins are HER3 and HER2;   normalizing the at least two cancer associated proteins in the sample to obtain a normalized value for each cancer associated protein in the sample;   dividing the normalized value of the first cancer associated protein with the normalized value of the second cancer associated protein to obtain a biomarker indicator comprising a ratio of the normalized value of HER3 to the normalized value of HER2; and   correlating the biomarker indicator with prognosis of the subject with cancer when the biomarker reaches a predetermined cut-off value obtained through recursive partitioning.   
     
     
         2 . The method of  claim 1 , wherein the subject is a human. 
     
     
         3 . The method of  claim 1 , wherein the subject is a non-human mammal. 
     
     
         4 . The method of  claim 1 , wherein the cancer is a solid tumor. 
     
     
         5 . The method of  claim 1 , wherein the at least two cancer associated proteins further comprises one or more tumor antigens selected from the group consisting of AKT; p-AKT; Blood Group Tn Antigen, CA150; CA19-9; CA50; CAB39L; CD22; CD24; CD63; CD66a+CD66c+CD66d+CD66e; CTAG1B; CTAG2; Carcino Embryonic Antigen (CEA); EBAG9; EGFR; FLJ14868; FMNL1; GAGE1; GPA33; Ganglioside OAcGD3; Heparanase 1; HER2; HER3; JAKMIP2; LRIG3; Lung carcinoma Cluster 2; M2A Oncofetal Antigen, MAGE 1; MAGEA10; MAGEA11; MAGEA12; MAGEA2; MAGEA4; MAGEB1; MAGEB2; MAGEB3; MAGEB4; MAGEB6; MAGEC1; MAGEE1; MAGEH1; MAGEL2; MGEA5; MOK protein kinase; MAPK; p-MAPK; mTOR; p-mTOR; MUC16; MUC4; Melanoma Associated Antigen; Mesothelin; Mucin 5AC; Neuroblastoma; OCIAD1; OIP5; Ovarian Carcinoma-associated Antigen; PAGE4; PCNA; PRAME; Plastin L; Prostate Mucin Antigen (PMA); Prostate Specific Antigen (PSA); PTEN; RASD2; ROPN1; SART2; SART3; SBEM; SDCCAG10; SDCCAG8; SPANX; SPANXB1; SSX5; STEAP4; STK31; TAG72; TEM1; XAGE2; Wilms' Tumor Protein, alpha 1 Fetoprotein; tumor antigens of epithelial origin; EGF, EGFR, Beta-catenin, Glycoprotein 130, PLCG1, Erbin, MUC1, Grb2, Heat shock protein 90 kDa alpha (cytosolic), member A1, DLG4, PIK3R2, PICK1 and SHC1. 
     
     
         6 . The method of  claim 1 , wherein quantifying comprises:
 transferring the cancer associated proteins from a tissue section to a stack of two or more membranes;   probing the stack of membranes with primary antibodies for detection of individual epitopes on the membranes;   detecting with fluorescent secondary antibodies the primary antibodies bound to individual epitopes on the membranes; and   quantifying the intensity of the fluorescent secondary antibodies.   
     
     
         7 . The method of  claim 1 , wherein quantifying comprises immunohistochemistry, laser capture micro dissection, “one dimensional” electrophoretic gel, a “two-dimensional” electrophoretic gel, mass-spectrometry, tissue micro array, multiplex tissue immunoblotting, or a combination of two or more thereof. 
     
     
         8 . The method of  claim 1 , wherein the sample is a tissue sample. 
     
     
         9 . The method of  claim 8 , 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. 
     
     
         10 . A method of using a ratiometric biomarker indicator obtained from a solid gastric tumor sample from a subject as an indicative of relative gastric cancer prognosis or survival rates for the subject comprising:
 obtaining a biomarker indicator, the biomarker indicator being obtained by a method comprising:
 extracting HER3 protein from the solid gastric tumor sample to produce a fraction comprising HER3 protein; 
 calculating the content of HER3 protein in the fraction; 
 normalizing the HER3 protein content against total cellular protein content in the sample 
 extracting HER2 protein from the solid tumor sample; 
 calculating the content of HER2 protein in the fraction; 
 normalizing the HER2 protein content against total cellular protein content in the sample; and 
 dividing the normalized HER3 protein content by the normalized HER2 protein content to obtain a ratiometric biomarker indicator, 
   wherein the biomarker indicator indicates prognosis or relative survival rate for the subject with the solid tumor.   
     
     
         11 . A method for using at least two cancer associated proteins as an indication of the presence of a gastric cancer in a subject, comprising:
 detecting in a gastric tissue sample from the subject at least two cancer associated proteins, wherein the first and second cancer associated proteins are HER3 and HER2;   calculating the level of protein expression for each of the HER3 and HER2 proteins; and   comparing the ratio of the level of protein expression for HER3 protein to the level of the protein expression for HER2 protein detected in the gastric tissue sample to a predetermined statistically significant cut-off value obtained through recursive partitioning, wherein a value of the ratio of protein expression of HER3 protein to HER2 protein in the gastric tissue sample below the cut-off value is indicative of the presence of gastric cancer in the subject.