Circulating tumor cell diagnostics for prostate cancer biomarkers

Abstract

The present invention describes a method for detecting castration-resistant prostate cancer (CRPC) in a patient afflicted with prostate cancer comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from the patient to detect circulating tumor cells (CTC), (b) determining prevalence of a CTC subpopulation associated with CRPC comprising detecting a measurable feature of each biomarker in a panel of morphological and protein biomarkers, and (c) comparing the prevalence of said CTC subpopulation to a predetermined threshold value, wherein the prevalence of the CTC subpopulation associated with CRPC above said predetermined threshold value is indicative of CRPC. In some embodiments, the CTC subpopulation associated with CRPC comprises CK− CTCs. In some embodiments, the CTC subpopulation associated with CRPC comprises small CTCs. In additional embodiments, the methods of the invention further comprise molecular analysis of the CTCs.

1 . A method for detecting castration-resistant prostate cancer (CRPC) in a patient afflicted with prostate cancer comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from the patient to detect circulating tumor cells (CTC), (b) determining prevalence of a CTC subpopulation associated with CRPC comprising detecting a measurable feature of each biomarker in a panel of morphological and protein biomarkers, and (c) comparing the prevalence of said CTC subpopulation to a predetermined threshold value, wherein the prevalence of the CTC subpopulation associated with CRPC above said predetermined threshold value is indicative of CRPC. 
     
     
         2 . The method of  claim 1 , wherein the CRPC is metastatic castration-resistant prostate cancer (mCRPC). 
     
     
         3 . The method of  claim 1 , further comprising an initial step of depositing the nucleated cells as a monolayer onto a slide. 
     
     
         4 . The method of  claim 1 , wherein the direct analysis comprises fluorescent scanning microscopy, optionally wherein the microscopy provides a field of view comprising CTCs and at least 200 surrounding white blood cells (WBCs). 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the CTCs comprise distinct morphological characteristics compared to surrounding nucleated cells, optionally wherein the morphological characteristics comprise one or more of the group consisting of nucleus size, nucleus shape, presence of holes in nucleus, cell size, cell shape and nuclear to cytoplasmic ratio, nuclear detail, nuclear contour, prevalence of nucleoli, quality of cytoplasm and quantity of cytoplasm. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the detection of CTCs further comprises comparing intensity of pan cytokeratin (CK) fluorescent staining to surrounding nucleated cells. 
     
     
         9 . The method of  claim 1 , further comprising an initial step of obtaining a white blood cell (WBC) count for the blood sample. 
     
     
         10 . The method of  claim 1 , further comprising an initial step of lysing erythrocytes in the blood sample. 
     
     
         11 . The method of  claim 1 , wherein the immunofluorescent staining of nucleated cells comprises pan cytokeratin (CK), cluster of differentiation (CD) 45, and diamidino-2-phenylindole (DAPI). 
     
     
         12 . The method of  claim 1 , wherein the immunofluorescent staining of nucleated cells further comprises Androgen Receptor (AR). 
     
     
         13 . The method of  claim 1 , wherein the CTC subpopulation associated with CRPC comprises CK− CTCs or small CTCs. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the direct analysis in step (a) detects CTCs selected from the group consisting of traditional CTCs, cytokeratin negative (CK − ) CTCs, small CTCs, and CTC clusters. 
     
     
         16 . The method of  claim 1 , wherein determining the presence of a CTC subpopulation associated with mCRPC in step (b) comprises analysis of the CTCs detected in step (a) at the single cell level. 
     
     
         17 . The method of  claim 1 , further comprising molecular characterization of the CTCs. 
     
     
         18 . The method of  claim 17 , wherein said molecular characterization comprises fluorescence in situ hybridization (FISH), wherein said FISH detects
 (a) rearrangement of erythroblast transformation-specific (ETS)-related gene (ERG) or   (b) loss of Phosphatase and tensin homolog gene (PTEN).   
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . A method for detecting progression of prostate cancer to CRPC in a patient comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characteristization of nucleated cells in a blood sample obtained from the patient to detect circulating tumor cells (CTC); (b) determining prevalence of a CTC subpopulation associated with CRPC comprising detecting a measurable feature of each biomarker in a panel of morphological and protein biomarkers, wherein the presence of the CTC subpopulation associated with CRPC is indicative of CRPC, and (c) repeating steps (a) and (b), wherein increase in the prevalence of the presence of the CTC population associated with CRPC indicates progression of prostate cancer to CRPC. 
     
     
         22 . The method of  claim 21 , wherein the CRPC is mCRPC. 
     
     
         23 . The method of  claim 22 , wherein said patient is undergoing androgen deprivation therapy (ADT). 
     
     
         24 . The method of  claim 21 , wherein the increase in prevalence is of a CTC subpopulation associated with CRPC comprises CK− CTCs or small CTCs. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 21 , wherein said increase in the prevalence of the CTC population associated with CRPC
 (a) predicts resistance to androgen deprivation therapy (ADT), or   (b) informs a subsequent decision to initiate secondary hormonal therapy directed at AR inhibition.   
     
     
         27 . (canceled)