Use of microvesicles in analyzing kras mutations

Abstract

Microvesicles are small membrane vesicles that either shed or bud off eukarotic cells. Analysis of the nucleic acid content of microvesicles may be useful in detecting the presence or absence of genetic aberrations. This invention discloses novel methods of diagnosing, prognosing, monitoring, or treating a disease, such as cancer, or other medical condition in a subject involving analyzing one or more nucleic acids contained within an isolated microvesicle for the presence or absence of one or more Kras genetic aberrations.

1 - 26 . (canceled) 
     
     
         27 . A method for diagnosis or prognosis of a disease or other medical condition associated with a Kras genetic aberration in a subject, comprising analyzing nucleic acid contained within microvesicles isolated from a body fluid of the subject, for the presence of a Kras genetic aberration. 
     
     
         28 . The method  claim 27 , wherein the microvesicles are treated with DNase prior to analysis to eliminate DNA located outside of the microvesicles. 
     
     
         29 . The method of  claim 27 , wherein the Kras genetic aberration is selected from the group consisting of G12A, G12D, G12R, G12C, G12S, G12V, and G13D. 
     
     
         30 . The method of  claim 27 , wherein the disease or other medical condition is cancer. 
     
     
         31 . The method of  claim 30 , wherein the cancer is selected from the group consisting of colorectal, pancreatic, thyroid, lung, acute myeloid leukemia, and glioblastoma. 
     
     
         32 . The method of  claim 27 , wherein the body fluid is blood, plasma, serum, urine, or combinations thereof. 
     
     
         33 . The method of  claim 27 , wherein the microvesicles are enriched for those originating from a specific cell type. 
     
     
         34 . The method of  claim 33 , wherein the specific cell type is lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, or fetus cells. 
     
     
         35 . The method of  claim 33 , wherein a microvesicular surface molecule is used to enrich for microvesicles from a specific cell type. 
     
     
         36 . The method of  claim 35 , wherein the microvesicular surface molecule is a surface antigen associated with tumor cells. 
     
     
         37 . The method of  claim 35 , wherein the microvesicular surface molecule is epithelial-cell-adhesion-molecule (EpCAM), CD24, CD70, carcinoembryonic antigen (CEA), EGFR, EGFRvIII and other variants, Fas ligand, TRAIL, transferrin receptor, p38.5, p97, or HSP72. 
     
     
         38 . The method of  claim 33 , wherein the absence of a microvesicular surface molecule is used to enrich for microvesicles from a specific cell type. 
     
     
         39 . The method of  claim 38 , wherein the surface molecule is CD80 or CD86. 
     
     
         40 . The method of  claim 33 , wherein the microvesicles are enriched using antibodies, aptamers, aptamer analogs, or molecularly imprinted polymers. 
     
     
         41 . The method of  claim 27 , wherein the nucleic acids are extracted from the microvesicles prior to analysis. 
     
     
         42 . The method of  claim 27 , wherein the nucleic acid is DNA. 
     
     
         43 . The method of  claim 27 , wherein the nucleic acid is RNA. 
     
     
         44 . The method of  claim 43 , wherein the RNA is reverse-transcribed into complementary DNA. 
     
     
         45 . The method of  claim 27 , wherein the nucleic acid is analyzed directly without an amplification step. 
     
     
         46 . The method  claim 27 , wherein the nucleic acid is amplified prior to analysis. 
     
     
         47 . The method of  claim 46 , wherein the nucleic acid is amplified by polymerase chain reaction (PCR), in situ PCR, quantitative PCR, nested PCR, self-sustained sequence replication, transcriptional amplification system, Qb Replicase, or cold-PCR. 
     
     
         48 . The method of  claim 27 , wherein the subject is a human colorectal cancer patient. 
     
     
         49 . A method for detecting a Kras genetic aberration in a subject, comprising analyzing nucleic acid contained within microvesicles isolated from a body fluid of a subject, for the presence of a Kras genetic aberration. 
     
     
         50 . A method for monitoring the status of a disease or other medical condition associated with a Kras genetic aberration in a subject, comprising periodically analyzing nucleic acid contained within microvesicles isolated from a body fluid of a subject, for the presence of a Kras genetic aberration associated with the disease or other medical condition. 
     
     
         51 . A method for evaluating treatment efficacy in a subject for a disease or other medical condition in which a Kras genetic aberration is associated with unresponsiveness to therapy, comprising analyzing nucleic acid contained within microvesicles isolated from a body fluid of the subject, for the presence of a Kras genetic aberration, wherein detection of the presence of a Kras genetic aberration indicates unresponsiveness to therapy.