Cell response assay for cancer and methods of producing and using same

Abstract

A cell response assay for cancer is provided. In the assay, the levels of a cancer cell type biomarker, a chemo resistance biomarker and a metastatic potential biomarker are simultaneously measured in a biological sample.

What is claimed is: 
     
         1 . A method of monitoring cancer treatment in a cancer patient undergoing said treatment, wherein the treatment is monitored using a multiplexed direct affinity assay, and wherein the method comprises the steps of:
 (A) isolating cells from a first biological sample taken from a cancer patient at a first time point;   (B) isolating cells from a second biological sample taken from a cancer patient at a second time point, wherein the cancer patient has been exposed to a cancer treatment between the first and second time points;   (C) reacting the isolated cells from the first biological sample with at least three affinity reagents for biomarkers, wherein the isolated cells are reacted with the at least three affinity reagents in a single reaction, wherein each affinity reagent comprises a different fluorescent label whereby the different fluorescent labels are measured at different excitations and emissions wavelengths, and wherein the at least three biomarkers comprise:
 (i) a cancer cell type biomarker; 
 (ii) a metastatic potential biomarker; and 
 (iii) a chemo resistance biomarker; 
 wherein biomarkers (i), (ii), and (iii) all co-exist in the cancer disease state, are fundamental to progression or relapse of the cancer disease state, and are all biochemically linked in a biochemical process; and 
 wherein the presence of markers (ii) and (iii) are linked such that the presence of one facilitates absence of the other; 
   (D) reacting the isolated cells from the second biological sample with the at least three affinity reagents of (C), wherein the isolated cells are reacted with the at least three affinity reagents in a single reaction;   (E) simultaneously measuring, in a multiplexed assay, expression levels of the at least three biomarkers in the isolated cells from the first biological sample;   (F) simultaneously measuring, in a multiplexed assay, the expression levels of the at least three biomarkers in the isolated cells from the second biological sample;   (G) calculating a proportion of cells detected by the cancer cell type biomarker that are also detected by the metastatic potential biomarker in the first and second biological samples;   (H) calculating a proportion of cells detected by the cancer cell type biomarker that are also detected by the chemo resistance biomarker in the first and second biological samples; and   (I) determining that the cancer patient is responding to treatment if:
 (1) the metastatic potential biomarker is present in a greater proportion of cancer cells detected by the cancer cell type biomarker in the second biological sample when compared to the first biological sample; and 
 (2) the chemo resistance biomarker is present in a lesser proportion of cancer cells detected by the cancer cell type biomarker in the second biological sample when compared to the first biological sample; or 
 determining that the cancer is progressing and/or relapsing if (1) and (2) are not present. 
   
     
     
         2 . The method of  claim 1 , wherein the cancer patient is suffering from a cancer selected from the group consisting of lung, bronchus, colon, rectum, pancreas, prostate, breast, liver, bile duct, bladder, ovary, brain, central nervous system (CNS), kidney, pelvis, uterine corpus, oral cavity, pharynx, melanoma, and combinations thereof. 
     
     
         3 . The method of  claim 1 , wherein the at least one cancer cell type biomarker is selected from the group consisting of epithelial cell adhesion molecule (EpCAM), a cytokeratin, vimentin, galectin-3, a cadherin, an oncoprotein, an oncogene, and combinations thereof. 
     
     
         4 . The method of  claim 3 , wherein the at least one cancer cell type biomarker comprises at least one of:
 (a) a combination of at least one type I cytokeratin and at least one type II cytokeratin;   (b) a combination of vimentin and galectin-3; and   (c) a combination of N-cadherin and E-cadherin.   
     
     
         5 . The method of  claim 3 , wherein the oncoprotein/oncogene is selected from the group consisting of HER2/neu, VEGF-165, KRAS, EGFr, WAF, BAX-1, PDGF, Rb, Jagged 1, Notch, VEGF, VEGHR, k-Ras, CAIX, MIB1, MDM, PR, ER, SEL5, SEM1, PI3K, Akt2, twist 1, EML-4, ALK, Braf, DRAFF, c-met, and combinations thereof. 
     
     
         6 . The method of  claim 1 , wherein at least one of:
 (a) the patient is suffering from prostate cancer, and the at least one cancer cell type biomarker comprises at least one of prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and combinations thereof;   (b) the patient is suffering from breast cancer, and the at least one cancer cell type biomarker comprises at least one of MUC1, CA 15-3, CA 27-29, and combinations thereof;   (c) the patient is suffering from colon cancer, and the at least one cancer cell type biomarker comprises at least one of Carcinoembryonic Antigen (CEA), CA19-9, Galactosyl Transferase II, and combinations thereof;   (d) the patient is suffering from pancreatic cancer, and the at least one cancer cell type biomarker comprises MSLN (mesothelin);   (e) the patient is suffering from ovarian cancer, and the at least one cancer cell type biomarker comprises at least one of CA 125, Follicle-Stimulating Hormone (FSH) receptor, and combinations thereof;   (f) the patient is suffering from liver cancer, and the at least one cancer cell type biomarker comprises Alpha-Fetoprotein;   (g) the patient is suffering from melanoma, and the at least one cancer cell type biomarker comprises at least one of Melan-A (MLANA), Tyrosinase (TYR), CSPG4, MITF and combinations thereof; and   (h) the patient is suffering from thyroid cancer, and the at least one cancer cell type biomarker comprises at least one of Parathyoid related protein (PTHP), TSHR, and combinations thereof.   
     
     
         7 . The method of  claim 1 , wherein the at least one chemo resistance biomarker comprises a cancer stem cell biomarker. 
     
     
         8 . The method of  claim 7 , wherein the at least one chemo resistance biomarker is selected from the group consisting of PL2L piwi like, ADLH, beta-integrin, alpha-6-integrin, c-kit, c-met, LIF-R, CXCR4, ESA, CD 20, CD44, CD133, CKS, TRAF2, ABC transporters, and combinations thereof. 
     
     
         9 . The method of  claim 7 , wherein the at least one chemo resistance biomarker comprises at least one of:
 (a) presence of CD44 and absence of CD24;   (b) presence of CD34 and absence of CD45 and CD31 (c) presence of CD44, CD24, and ESA; and   (c) presence of CD24 and ESA.   
     
     
         10 . The method of  claim 1 , wherein the at least one metastatic potential biomarker is selected from the group consisting of urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), CD95, a serine protease, a serine protease inhibitor, a matrix metalloproteinase, a matrix metalloproteinase inhibitor, and combinations thereof. 
     
     
         11 . The method of  claim 10 , wherein at least one of:
 (a) the serine protease is selected from the group consisting of plasmin, ADAM, and combinations thereof;   (b) the serine protease inhibitor comprises Bikunin;   (c) the matrix metalloproteinase comprises MMP9; and   (d) the matrix metalloproteinase inhibitors comprises TIMP-1.   
     
     
         12 . The method of  claim 1 , wherein at least one of the affinity reagents comprises a conjugate of a fluorescent label to an antibody to the biomarker. 
     
     
         13 . The method of  claim 1 , wherein the labels of the first, second, and third labeled probes are selected from the group consisting of fluorescein-5-isothiocyanate (FITC), phycoerythrin, sulforhodamine 101(Texas Red), 2-[4-(aminoiminomethyl)phenyi]-1H-Indole-6-carboximidamide (DAPI), 3H-Indolium (Cy5), 1H-benz[e]indolium (Cy 5.5), 3H-Indolium (Cy 7), ALEXA FLUOR® 488, ALEXA FLUOR® 555, ALEXA FLUOR® 647, rare earth metals, rare earth element-containing nanoparticles, and combinations and derivatives thereof. 
     
     
         14 . The method of  claim 1 , wherein the biological sample is further defined as a tissue sample. 
     
     
         15 . The method of  claim 1 , wherein at least four biomarkers are simultaneously measured, wherein the fourth biomarker comprises at least one of:
 (a) at least one white blood cell biomarker selected from the group consisting of CD45, CTLA-4, CD4, CD68, CD8, and combinations thereof, and wherein the method further comprises the step of excluding cells positive for at least one white blood cell biomarker from the calculating steps;   (b) a biomarker indicating the presence of cell nuclei, and wherein the fourth labeled probe comprises 4′,6-diamidino-2′-phenylindole, dihydrochloride (DAPI); and   (c) phosphatidylserine, and wherein the fourth labeled probe comprises at least one of bis(zinc 2 +dipicolylamine) and PSVue™.   
     
     
         16 . The method of  claim 1 , wherein the method is further defined as a method of monitoring breast cancer treatment, and wherein biomarker (i) is HER2/neu, biomarker (ii) is uPA and PAI-1, and biomarker (iii) is PL2L piwi like. 
     
     
         17 . The method of  claim 1 , wherein the method is further defined as a method of monitoring prostate cancer treatment, and wherein biomarker (i) is PSA, biomarker (ii) is uPA and PAI-1, and biomarker (iii) is PL2L piwi like. 
     
     
         18 . A method of monitoring cancer treatment in a cancer patient undergoing said treatment, wherein the treatment is monitored using a multiplexed direct affinity assay, and wherein the method comprises the steps of:
 (A) isolating cells from a first biological sample taken from a cancer patient at a first time point;   (B) isolating cells from a second biological sample taken from a cancer patient at a second time point, wherein the cancer patient has been exposed to a cancer treatment between the first and second time points;   (C) reacting the isolated cells from the first biological sample with at least three affinity reagents for biomarkers, wherein the isolated cells are reacted with the at least three affinity reagents in a single reaction, wherein each affinity reagent comprises a different fluorescent label whereby the different fluorescent labels are measured at different excitations and emissions wavelengths, and wherein the at least three biomarkers comprise:
 (i) a cancer cell type biomarker; 
 (ii) a metastatic potential biomarker; and 
 (iii) a chemo resistance biomarker; 
 wherein biomarkers (i), (ii), and (iii) all co-exist in the cancer disease state, are fundamental to progression or relapse of the cancer disease state, and are all biochemically linked in a biochemical process; and 
 wherein the presence of markers (ii) and (iii) are linked such that the presence of one facilitates absence of the other; 
   (D) reacting the isolated cells from the second biological sample with the at least three affinity reagents of (C), wherein the isolated cells are reacted with the at least three affinity reagents in a single reaction;   (E) simultaneously measuring, in a multiplexed assay, expression levels of the at least three biomarkers in the isolated cells from the first biological sample;   (F) simultaneously measuring, in a multiplexed assay, the expression levels of the at least three biomarkers in the isolated cells from the second biological sample;   (G) calculating a proportion of cells detected by the cancer cell type biomarker that are also detected by the metastatic potential biomarker in the first and second biological samples;   (H) calculating a proportion of cells detected by the cancer cell type biomarker that are also detected by the chemo resistance biomarker in the first and second biological samples; and   (I) administering a subsequent dose of the cancer treatment if:
 (1) the metastatic potential biomarker is present in a greater proportion of cancer cells detected by the cancer cell type biomarker in the second biological sample when compared to the first biological sample; and 
 (2) the chemo resistance biomarker is present in a lesser proportion of cancer cells detected by the cancer cell type biomarker in the second biological sample when compared to the first biological sample. 
   
     
     
         19 . The method of  claim 18 , wherein the method is further defined as a method of monitoring breast cancer treatment, and wherein biomarker (i) is HER2/neu, biomarker (ii) is uPA and PAI-1, and biomarker (iii) is PL2L piwi like. 
     
     
         20 . The method of  claim 18 , wherein the method is further defined as a method of monitoring prostate cancer treatment, and wherein biomarker (i) is PSA, biomarker (ii) is uPA and PAI-1, and biomarker (iii) is PL2L piwi like.