Circulating tumor cell assay

Abstract

Methods for the detection, enumeration and analysis of circulating tumor cells expressing insulin-like growth factor-1 receptors (IGF-1R) are disclosed. These methods are useful for cancer screening and staging, development of treatment regimens, and for monitoring for treatment responses, cancer recurrence or the like. Test kits that facilitate the detection, enumeration and analysis of such circulating tumor cells are also provided.

What is claimed is: 
     
         1 . A method for predicting efficacy of IGF-1R antagonist therapy in a patient, comprising the steps of:
 a) preparing a sample wherein a blood sample from the patient is mixed with a ligand that reacts specifically with tumor cells, to the substantial exclusion of other sample components, so as to obtain a cell population enriched for tumor cells;   b) contacting the enriched cell population with an anti-cytokeratin antibody;   c) contacting the enriched cell population with an antibody to insulin-like growth factor receptors (IGF-1R); and   d) determining the presence of cells bound by both the anti-cytokeratin antibody and the anti-IGF-1R antibody;   wherein the presence of cells bound by both antibodies is predictive of efficacy of IGF-1R antagonist therapy in the patient.   
     
     
         2 . The method of  claim 1 , wherein the determining step comprises determining the number of cells bound by both antibodies. 
     
     
         3 . The method of  claim 1 , further comprising the step of adding to the sample a cell specific dye to allow exclusion of residual non-nucleated cells and cell debris from analysis. 
     
     
         4 . The method of  claim 1 , wherein the determining step is performed using at least one process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, CELLSPOTTER analysis, and automated cell analysis. 
     
     
         5 . The method of  claim 1 , wherein the sample is an immunomagnetic sample comprising the blood sample mixed with magnetic particles coupled to the ligand, and further comprising the step of subjecting the immunomagnetic sample to a magnetic field, such that the immunomagnetic sample becomes an enriched tumor cell suspension. 
     
     
         6 . The method of  claim 5 , wherein the magnetic particles are colloidal. 
     
     
         7 . A method for monitoring efficacy of an IGF-1R antagonist therapy-in a patient, comprising the steps of:
 a) preparing a first sample wherein a first blood sample from the patient is mixed with a ligand that reacts specifically with tumor cells, to the substantial exclusion of other sample components, so as to obtain a cell population enriched for tumor cells;   b) contacting the enriched cell population with an anti-cytokeratin antibody;   c) contacting the enriched cell population with an antibody to insulin-like growth factor receptors (IGF-1R);   d) determining the presence and number of cells bound by both the anti-cytokeratin antibody and the anti-IGF-1R antibody;   e) preparing a second sample from a second blood sample from the patient, after administration of an IGF-1R antagonist therapy, wherein the second blood sample is mixed with the ligand that reacts specifically with tumor cells, and performing steps b)-d) on the second sample; and   f) comparing the number of cells bound by both the anti-cytokeratin antibody and the anti-IGF-1R antibody in the first sample to the number of cells bound by both antibodies in the second sample;   wherein a lower number of cells bound by both antibodies in the second sample is indicative of efficacy of the IGF-1R antagonist therapy in the patient.   
     
     
         8 . The method of  claim 7 , wherein the determining steps are performed using at least one process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, CELLSPOTTER analysis, and automated cell analysis. 
     
     
         9 . The method of  claim 7 , wherein the sample is an immunomagnetic sample comprising the blood sample mixed with magnetic particles coupled to the ligand, and further comprising the step of subjecting the immunomagnetic sample to a magnetic field, such that the immunomagnetic sample becomes an enriched tumor cell suspension. 
     
     
         10 . The method of  claim 9 , wherein the magnetic particles are colloidal. 
     
     
         11 . The method of  claim 1 , wherein the IGF-1R antagonist therapy comprises an anti-IGF-1R antibody. 
     
     
         12 . The method of  claim 7 , wherein the IGF-1R antagonist therapy comprises an anti-IGF-1R antibody. 
     
     
         13 . The method of  claim 1 , wherein the anti-cytokeratin antibody is labeled with fluorescein isothiocyanate (FITC). 
     
     
         14 . The method of  claim 1 , wherein the antibody to IGF-1R is labeled with phycoerythrin (PE). 
     
     
         15 . The method of  claim 1 , wherein the ligand is an antibody to epithelial cell adhesion molecule (EpCAM). 
     
     
         16 . The method of  claim 7 , wherein the anti-cytokeratin antibody is labeled with fluorescein isothiocyanate (FITC). 
     
     
         17 . The method of  claim 7 , wherein the antibody to IGF-1R is labeled with phycoerythrin (PE). 
     
     
         18 . The method of  claim 7 , wherein the ligand is an antibody to epithelial cell adhesion molecule (EpCAM). 
     
     
         19 . A test kit for screening a patient sample for the presence of circulating tumor cells expressing IGF-1R, comprising:
 a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a characteristic determinant of the tumor cells, the antibody being coupled, directly or indirectly, to said base coating material;   b) a cell specific dye for excluding sample components other than the tumor cells from analysis; and   c) at least one detectably labeled agent having binding affinity for IGF-1R.   
     
     
         20 . The test kit of  claim 19 , further comprising a device for the detection of circulating tumor cells expressing IGF-1R.