Digital Analysis of Circulating Tumor Cells in Blood Samples

Abstract

This disclosure relates to new assay methods for analysis of circulating tumor cells (CTCs) in blood samples for detection, e.g., early detection, and/or monitoring of disease, e.g., cancer. The methods provide ultra-high sensitivity and specificity, and include the use of microfluidic isolation of CTCs and digital detection of RNA derived from the CTCs.

1 . A method for analyzing circulating tumor cells (CTCs) in a blood sample with ultra-high sensitivity and specificity, the method comprising
 isolating from a blood sample a product comprising CTCs and other cells present in blood;   isolating ribonucleic acid (RNA) molecules from the product   generating cDNA molecules in solution from the isolated RNA;   encapsulating cDNA molecules into individual droplets;   amplifying cDNA molecules in each droplet in the presence of [a] one or more reporter groups configured to bind specifically to cDNA from CTCs and not to cDNA from other cells in the blood;   detecting droplets that contain bound reporter groups as an indicator of the presence of cDNA molecules from CTCs in the droplets; and   analyzing CTCs in the detected droplets.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , further comprising reducing a volume of the product before isolating RNA. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein generating cDNA molecules from the isolated RNA comprises conducting reverse transcription (RT) polymerase chain reaction (PCR) of the isolated RNA molecules. 
     
     
         6 . The method of  claim 1 , wherein amplifying cDNA or cDNA molecules within each of the droplets comprises conducting PCR in each droplet. 
     
     
         7 . The method of  claim 1 , wherein encapsulating individual cDNA molecules further comprises encapsulating PCR reagents in individual droplets with the cDNA molecules and forming at least 1000 droplets of a non-aqueous liquid. 
     
     
         8 . The method of  claim 1 , wherein the one or more reporter groups comprise a fluorescent label. 
     
     
         9 . The method of  claim 4 , wherein removing contaminants from the cDNA-containing solution comprises the use of Solid Phase Reversible Immobilization (SPRI), comprising immobilizing cDNA in the solution with magnetic beads that are configured to specifically bind to the cDNA; removing contaminants from the solution; and eluting purified cDNA. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 7 , wherein the non-aqueous liquid comprises one or more fluorocarbons, hydrofluorocarbons, mineral oils, silicone oils, and hydrocarbon oils. 
     
     
         12 . The method of  claim 6 , wherein probes and primers for use in amplifying the cDNA molecules within each of the droplets correspond to one or more probes and primers that relate to one or more selected cancer-selective genes listed in Table 1. 
     
     
         13 . The method of  claim 12 , wherein the selected cancer-selective genes include prostate cancer-selective genes. 
     
     
         14 . The method of  claim 12 , wherein the selected cancer-selective genes include breast cancer-selective genes. 
     
     
         15 . The method of  claim 12 , wherein the selected cancer-selective genes include genes selective for one or more of lung cancer, pancreatic cancer, liver cancer, and melanoma. 
     
     
         16 . The method of  claim 12 , wherein the selected cancer-selective genes include one or more genes selective for two or more, three or more, four or more, or five or more different types of cancer. 
     
     
         17 . The method of  claim 16 , wherein the genes are selective for breast cancer and lung cancer; breast cancer, lung cancer, and liver cancer; breast cancer, lung cancer, and pancreatic cancer; breast cancer, lung cancer, and prostate cancer; breast cancer, liver cancer, and melanoma; breast cancer, lung cancer, and melanoma; breast cancer, lung cancer, liver cancer, and prostate cancer; breast cancer, lung cancer, liver cancer, and melanoma; breast cancer, lung cancer, liver cancer, and pancreatic cancer; breast cancer, lung cancer, prostate cancer, and pancreatic cancer; breast cancer, lung cancer, liver cancer, melanoma, and pancreatic cancer; or breast cancer, lung cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer. 
     
     
         18 . The method of  claim 1 , wherein the CTCs arise from metastatic or primary/localized cancers. 
     
     
         19 . The method of  claim 1 , wherein analyzing the CTCs in the detected droplets comprises monitoring CTCs from blood samples taken over time from a patient with a known cancer, and testing, imaging, or both testing and imaging the CTCs to provide a prognosis for the patient. 
     
     
         20 . The method of  claim 1 , wherein analyzing the CTCs in the detected droplets comprises testing, imaging, or testing and imaging the CTCs from a blood sample from a patient to provide an indication of a response by the CTCs to a therapeutic intervention. 
     
     
         21 . The method of  claim 1 , wherein analyzing the CTCs in the detected droplets comprises determining a number or level of CTCs per unit volume of a blood sample from a patient to provide a measure of tumor burden in the patient. 
     
     
         22 . The method of  claim 21 , further comprising using the measure of tumor burden in the patient to select a therapy. 
     
     
         23 . The method of  claim 22 , further comprising determining the measure of tumor burden in the patient at a second time point to monitor the tumor burden over time. 
     
     
         24 - 28 . (canceled)