Apparatus and method for absolute quantification of biomarkers for solid tumor diagnosis

Abstract

Provided is a method for quantitative analysis of a sample. The method includes steps of (a) providing a singular marker representative of one or more features of the sample, the sample comprising a population of individual units of the marker; (b) measuring the marker with dot blot analysis, wherein the quantitation result is an absolute amount of the marker's population of individual units in the sample, normalized by the sample volume or by the sample weight; and (c) obtaining an objective determination of the one or more features of the sample based on the quantitation result of the marker. Also disclosed is a reference database and a method to use the reference database for diagnosing cancer in a patient.

1 . A method for quantitative analysis of a sample, comprising:
 (a) providing a singular marker representative of one or more features of the sample, the sample being a formalin-fixed paraffin-embedded (FFPE) tissue sample and comprising a population of individual units of the marker;   (b) quantifying the marker extracted from the FFPE tissue sample with dot blot analysis, wherein the quantitation result is an absolute amount of the marker's population of individual units in the sample, normalized by the protein weight; and   (c) obtaining a determination of the one or more features of the sample based on the quantitation result of the marker.   
     
     
         2 . The method of  claim 1 , wherein the step (b) comprises measuring the marker with quantitative dot blot (QDB) analysis. 
     
     
         3 . The method of  claim 2 , wherein the step (b) comprises:
 (b1) incubating the sample with a solution comprising a binding agent, wherein:   the binding agent is capable of specifically binding to an individual unit of the marker, and   the binding agent is capable of being quantified by the dot blot analysis; and   (b2) measuring the marker through QDB analysis of the binding agent.   
     
     
         4 . The method of  claim 3 , wherein the marker is a protein marker and the binding agent is an antibody. 
     
     
         5 . The method of  claim 4 , wherein the antibody is an analyte specific regent (ASR) antibody. 
     
     
         6 . The method of  claim 4 , wherein the antibody is an in vitro diagnostics (IVD) antibody. 
     
     
         7 . The method of  claim 4 , wherein the antibody is capable of being assayed in immunohistochemistry. 
     
     
         8 . The method of  claim 1 , wherein the sample is a biopsy tissue from a subject. 
     
     
         9 . The method of  claim 8 , wherein the subject is a cancer patient. 
     
     
         10 . The method of  claim 9 , further comprising (d) evaluating the patient based on the quantitation result of the marker. 
     
     
         11 . The method of  claim 10 , wherein, in step (d), evaluating the patient comprises diagnosis and prognosis of a cancer for the patient. 
     
     
         12 . The method of  claim 11 , wherein the diagnosis and prognosis of a cancer comprises disease-free survival, overall survival, hazard ratio, or treatment prediction for the cancer. 
     
     
         13 . The method of  claim 10 , wherein, in step (d), evaluating the patient is conducted by a commercial kit. 
     
     
         14 . A reference database for diagnosing cancer in a patient based on antibody-based quantitative analysis of a marker in a formalin-fixed paraffin-embedded_(FFPE) biopsy sample from the patient, comprising a plurality of reference profiles, each of the plurality of the reference profile prepared by:
 (a) providing a formalin-fixed paraffin-embedded (FFPE)_biopsy sample from a cancer patient with a known diagnosis;   (b) measuring said marker with dot blot analysis for the biopsy sample, wherein the quantitation result is an absolute amount of said marker in the biopsy sample, normalized by the protein weight thereof; and   (c) associating the quantitation result with the known diagnosis of the cancer patient thereby obtaining a reference profile.   
     
     
         15 . The reference database of  claim 14 , wherein the step (b) comprises:
 (b1) incubating the biopsy sample with a solution comprising a binding agent that specifically binding to said marker; and   (b2) measuring said marker through quantitative dot blot (QDB) analysis of the binding agent.   
     
     
         16 . The reference database of  claim 15 , wherein the marker is a protein marker and the binding agent is an antibody. 
     
     
         17 . A method for diagnosing cancer in a patient, comprising:
 providing a reference database of  claim 14 ;   obtaining a biopsy sample from the patient;   measuring the marker used in the reference database in the biopsy sample with QDB analysis, wherein the measured result is an absolute amount of the marker in the biopsy sample;   comparing the quantitation result of the marker with that of each reference profile stored in the reference database; and   identifying a reference profile in the reference database that has the best match and outputting the known diagnosis associated with the identified reference profile.   
     
     
         18 . The method of  claim 17 , wherein the cancer is a solid tumor cancer. 
     
     
         19 . A kit for diagnosing cancer in a patient, the kit comprising the reference database of  claim 14 . 
     
     
         20 . A kit for diagnosing cancer in a patient, the kit comprising a unit configured to access online the reference database of  claim 14 .