Detection of saliva proteins modulated secondary to ductal carcinoma in situ of the breast

Abstract

A method of differentiating among the presence of ductal carcinoma in situ in the breast, benign fibroadenoma of the breast, and non-cancerous breast tissue in a subject is disclosed. The method comprises: measuring the concentration of at least one protein biomarker selected from a group of forty-nine differentially expressed proteins in the saliva of persons with DCIS, or benign fibroadenoma, or in persons who are cancer-free. The resulting test data is compared to a reference panel. From the comparison the presence in the subject of either ductal carcinoma in situ of the breast, or benign fibroadenoma of the breast is determined.

1 . A method of diagnosing the likelihood of the presence or occurrence of breast tumor in a test subject, the method comprising:
 (a) measuring in a saliva sample from the test subject the concentration of at least a first protein biomarker, wherein each said biomarker is known to be differentially expressed in breast tumor and tumor-free breast tissue, wherein said breast tumor comprises benign fibroadenoma or ductal carcinoma in situ of the breast (DCIS), to provide a set of test data comprising a concentration value of each said protein biomarker in said saliva sample;   (b) comparing said test values to a reference panel comprising
 (1) a mean concentration value of each said protein in saliva from a group of breast tumor-free individuals (reference control group), 
 (2) a mean concentration value of each said protein in saliva from a group of individuals with DCIS (reference DCIS group), or from a group of individuals with benign fibroadenoma of the breast (reference benign group), or from both the DCIS group and the benign group; 
   (c) determining from said comparison a diagnosis of likelihood of the presence or occurrence of a breast tumor in the test subject.   
     
     
         2 . The method of  claim 1 , wherein
 in (a), at least said first biomarker is known to be differentially expressed among benign fibroadenoma, DCIS and tumor-free breast tissue;   in (b), (2) comprises:
 (2′) a mean concentration value of each said protein in saliva from said reference DCIS group, and 
 (2″) a mean concentration value of each said protein in saliva from said reference benign group, and 
   in (c), said determining comprises determining from said comparison a diagnosis of likelihood of the presence or occurrence of either DCIS or fibroadenoma of the breast in the test subject.   
     
     
         3 . The method of  claim 2 , wherein the concentration value of at least said first protein biomarker in said reference panel is significantly different in the saliva of said reference DCIS group and/or in the saliva of said reference benign group, relative to the respective level of each said protein biomarker in the saliva of said reference control group. 
     
     
         4 . The method of  claim 3 , wherein the concentration value difference of at least said first protein biomarker is significant at a level in the range of p<0.05 to p<0.0001. 
     
     
         5 . The method of  claim 3 , wherein at least said first protein biomarker is selected from the group consisting of CAH6, K2C4, CYTA, FABP4, IGHGI, TRFL, BPIL1, CYTC, HPT, PROF1 and ZA2G, and, in (c), said determining comprises determining from said comparison a diagnosis of likelihood of the presence or occurrence of DCIS in the test subject. 
     
     
         6 . The method of  claim 3 , wherein at least said first protein biomarker is selected from the group consisting of ENOA, IGHA2, IL-1ra, S10A7, SPLC2, and, in (c), said determining comprises determining from said comparison a diagnosis of likelihood of the presence or occurrence of fibroadenoma in the test subject. 
     
     
         7 . The method of  claim 1 , wherein said reference panel is prepared by analyzing salivary proteins by isotopic labeling and liquid chromatography tandem mass spectrometry to characterize salivary proteins from each said group, and determining from said analysis the differential concentrations of said protein biomarkers in individuals with ductal carcinoma in situ of the breast, in individuals with benign fibroadenoma of the breast, and in individuals free of both ductal carcinoma in situ and benign fibroadenoma. 
     
     
         8 . The method of  claim 1 , wherein, in (a), said measuring comprises analyzing said sample comprising salivary proteins by isotopic labeling and liquid chromatography tandem mass spectrometry to characterize said proteins. 
     
     
         9 . The method of  claim 8 , wherein said isotopic labeling comprises differential isotopic labeling of salivary proteins of at least first and second saliva samples. 
     
     
         10 . The method of  claim 1 , wherein, in (b), said comparing yields a comparison result in which the concentration value of at least said first protein biomarker is a significant change relative to the mean concentration value of the respective protein in the control group of the reference panel. 
     
     
         11 . The method of  claim 2 , wherein said saliva sample is a first saliva sample from said test subject, and, in (a), the set of test data is a first set of test data, and the method further comprises:
 (d) obtaining a second saliva sample from said subject subsequent to said first sample;   (e) measuring the concentration of at least said first protein biomarker in the second saliva sample, to provide a second set of test data comprising a second concentration value of each said protein biomarker in said saliva sample;   (f) comparing the second set of test data to said reference panel;   (g) determining from the result of said comparing a diagnosis of likelihood of the presence or occurrence of either DCIS or benign fibroadenoma of the breast in the test subject.   
     
     
         12 . The method of  claim 11 , further comprising (h) comparing said second set of test data to said first set of test data to determine whether a difference in the concentration value of at least said first protein biomarker exists between said first and second sets of test data for said test subject. 
     
     
         13 . The method of  claim 12 , wherein said first saliva sample is obtained prior to surgical removal of cancerous breast tissue from said subject. 
     
     
         14 . The method of  claim 13 , wherein said test subject has received therapeutic treatment for breast cancer prior to obtaining said second saliva sample. 
     
     
         15 . The method of  claim 14  wherein, in (g), determining a comparative decrease in at least said first protein biomarker concentration in said second sample relative to said first sample indicates that the therapeutic treatment is effective.