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.

1 . An assay for cancer, comprising the steps of:
 measuring at least one cancer cell type biomarker in a biological sample of a cancer patient utilizing a first labeled probe that binds to said cancer cell type biomarker;   measuring at least one chemo resistance biomarker in the biological sample utilizing a second labeled probe that binds to said chemo resistance biomarker;   measuring at least one metastatic potential biomarker in the biological sample utilizing a third labeled probe that binds to said metastatic potential biomarker; and   wherein the at least three labeled probes are measured at different excitations and emission wavelengths.   
     
     
         2 . The assay of  claim 1 , wherein the assay is for 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 assay 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 assay 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 assay 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 assay of  claim 1 , wherein at least one of:
 (a) the assay is for 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 assay is for 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 assay is for 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 assay is for pancreatic cancer, and the at least one cancer cell type biomarker comprises MSLN (mesothelin);   (e) the assay is for 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 assay is for liver cancer, and the at least one cancer cell type biomarker comprises Alpha-Fetoprotein;   (g) the assay is for 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 assay is for 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 assay of  claim 1 , wherein the at least one chemo resistance biomarker comprises a cancer stem cell biomarker. 
     
     
         8 . The assay of  claim 7 , wherein the at least one chemo resistance biomarker is selected from the group consisting of PL2L piwi like, ADLH, β-integrin, α6 integrin, c-kit, c-met, LIF-R, CXCR4, ESA, CD 20, CD44, CD133, CK5, TRAF2, ABC transporters and combinations thereof. 
     
     
         9 . The assay 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   (d) presence of CD24 and ESA.   
     
     
         10 . The assay 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 assay 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 assay of  claim 1 , wherein at least one of the first, second and third labeled probes comprise at least one labeled antibody to a biomarker. 
     
     
         13 . The assay 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)phenyl]-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 assay of  claim 1 , further comprising the step of isolating cancer cells from the biological sample prior to conducting the measuring steps, and wherein the measuring steps are further defined as measuring at least one cancer cell type biomarker, at least one chemo resistance biomarker and at least one metastatic potential biomarker in the cancer cells isolated from the biological sample. 
     
     
         15 . The assay of  claim 1 , wherein the biological sample is further defined as a tissue sample. 
     
     
         16 . The assay of  claim 1 , further comprising measuring at least one additional biomarker in the biological sample utilizing a fourth labeled probe that binds to said additional biomarker. 
     
     
         17 . The assay of  claim 16 , wherein the at least one additional biomarker comprises 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 assay further comprises the step of excluding cells positive for at least one white blood cell biomarker. 
     
     
         18 . The assay of  claim 16 , wherein the at least one additional biomarker comprises a biomarker indicating the presence of cell nuclei, and wherein the fourth labeled probe comprises 4′,6-diamidino-2′-phenylindole, dihydrochloride (DAPI). 
     
     
         19 . The assay of  claim 16 , wherein the at least one additional biomarker comprises phosphatidylserine, and wherein the fourth labeled probe comprises at least one of bis(zinc 2+ dipicolylamine) and PSVue™. 
     
     
         20 . A kit, comprising:
 a first labeled probe that binds to a cancer cell type biomarker;   a second labeled probe that binds to a chemo resistance biomarker;   a third labeled probe that binds to a metastatic potential biomarker; and   wherein the at least three labeled probes are measured at different excitations and emission wavelengths.   
     
     
         21 - 38 . (canceled) 
     
     
         39 . A method of monitoring cancer treatment in a cancer patient undergoing said treatment, the method comprising the steps of:
 measuring the levels of a cancer cell type biomarker in a first biological sample and in a second biological sample utilizing a first labeled probe that binds to said cancer cell type biomarker, wherein the first biological sample is obtained from the patient prior to exposure to a cancer treatment, and wherein the second biological sample is obtained from the patient following exposure to the cancer treatment;   measuring the levels of a chemo resistance biomarker in the first and second biological samples utilizing a second labeled probe that binds to said chemo resistance biomarker;   measuring the levels of a metastatic potential biomarker in the first and second biological samples utilizing a third labeled probe that binds to said metastatic potential biomarker, and wherein the at least three labeled probes are measured at different excitations and emission wavelengths;   comparing the levels of the chemo resistance biomarker in the cells to which the first labeled probe is bound in the first and second biological samples;   comparing the levels of the metastatic potential biomarker in the cells to which the first labeled probe is bound in the first and second biological samples; and   determining that the cancer treatment is effective if the level of the chemo resistance biomarker is decreased and the level of the metastatic potential biomarker is increased in the second biological sample when compared to the first biological sample.   
     
     
         40 . A method of monitoring progression/relapse of cancer in a cancer patient, the method comprising the steps of:
 measuring the levels of a cancer cell type biomarker in a first biological sample and in a second biological sample utilizing a first labeled probe that binds to said cancer cell type biomarker, wherein the first biological sample is obtained from the patient at a first time point, and the second biological sample is obtained from the patient at a subsequent time point;   measuring the levels of a chemo resistance biomarker in the first and second biological samples utilizing a second labeled probe that binds to said chemo resistance biomarker;   measuring the levels of a metastatic potential biomarker in the first and second biological samples utilizing a third labeled probe that binds to said metastatic potential biomarker, and wherein the at least three labeled probes are measured at different excitations and emission wavelengths;   comparing the levels of the chemo resistance biomarker in the cells to which the first labeled probe is bound in the first and second biological samples;   comparing the levels of the metastatic potential biomarker in the cells to which the first labeled probe is bound in the first and second biological samples; and   determining that the cancer has progressed/relapsed if the level of the chemo resistance biomarker is increased and the level of the metastatic potential biomarker is decreased in the second biological sample when compared to the first biological sample.   
     
     
         41 - 60 . (canceled)