US20130029339A1 - Use of microvesicles in analyzing kras mutations - Google Patents

Use of microvesicles in analyzing kras mutations Download PDF

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US20130029339A1
US20130029339A1 US13/395,354 US201013395354A US2013029339A1 US 20130029339 A1 US20130029339 A1 US 20130029339A1 US 201013395354 A US201013395354 A US 201013395354A US 2013029339 A1 US2013029339 A1 US 2013029339A1
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microvesicles
nucleic acid
kras
disease
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Johan Karl Olov Skog
Xandra O. Breakefield
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General Hospital Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the fields of medical diagnosis, and prognosis, patient monitoring, treatment efficacy, and molecular diagnostics based on the analysis of Kras nucleic acids extracted from microvesicles.
  • Molecular diagnostics used to diagnose, monitor, treat, and evaluate diseases and other medical conditions, is becoming an increasingly important tool, particularly with the accumulating knowledge of the molecular mechanisms underlying various types of diseases and medical conditions. Molecular diagnostics is particularly valuable in the context of cancer, since our knowledge of the underlying genetic causes of cancers is rapidly expanding.
  • Cancers arise through accumulation of genetic alterations that promote unrestricted cell growth. It has been stated that each tumor harbors, on average, around 50-80 mutations that are absent in non-tumor cells (Jones et al., 2008; Parsons et al., 2008; Wood et al., 2007).
  • One family of oncogenes that is commonly mutated in cancers is the RAS family.
  • Ras has been shown to play an important role in the expression of matrix metalloproteinases, as well as other processes that promote tumor invasion and metastasis.
  • the members of the Ras family which includes the Hras, Kras, and Nras genes, Kras is most commonly mutated in cancers.
  • This invention discloses novel methods of diagnosing, prognosing, monitoring, and treating a disease, such as cancer, or other medical condition in a subject involving the analysis of one or more nucleic acids contained within one or more microvesicles isolated from a bodily fluid sample for the presence or absence of one or more Kras genetic aberrations.
  • One aspect of the invention are methods for detecting the presence or absence of a Kras genetic aberration in a fluid sample, the methods comprising the steps of: (a) isolating one or more microvesicles from a fluid sample; and (b) analyzing one or more nucleic acids contained within the one or more microvesicles for the presence or absence of a Kras genetic aberration.
  • Another aspect of the invention are diagnostic or prognostic methods, wherein said methods aid in the diagnosis or prognosis of a disease or other medical condition in a subject, the methods comprising the steps of: (a) isolating one or more microvesicles from a body fluid sample from the subject; and (b) analyzing one or more nucleic acids contained within the one or more microvesicles for the presence or absence of a Kras genetic aberration associated with the diagnosis or prognosis of a disease or other medical condition.
  • a further aspect of the invention are monitoring methods, wherein said methods aid in monitoring the status of a disease or other medical condition in a subject over time, the methods comprising the steps of: (a) isolating one or more microvesicles from a body fluid sample from the subject; (b) analyzing one or more nucleic acids contained within the one or more microvesicles for the presence or absence of a Kras genetic aberration associated with the disease or other medical condition; and (c) repeating steps (a) and (b) after the passage of an interval of time.
  • Another aspect of the invention are evaluation methods, wherein said methods aid in evaluating treatment efficacy in a subject having a disease or other medical condition, the methods comprising the steps of: (a) isolating one or more a microvesicles from a body fluid sample from the subject; and (b) analyzing one or more nucleic acids contained within the one or more microvesicles for the presence or absence of a Kras genetic aberration associated with treatment efficacy for the disease or other medical condition.
  • the methods may further comprise the step of treating the one or more isolated microvesicles with DNase prior to analysis to eliminate all or substantially all of any DNA located on the surface of the one or more microvesicles or outside of the one or more microvesicles.
  • the Kras genetic aberration is selected from the group consisting of: G12A, G12D, G12R, G12C, G12S, G12V, or G13D.
  • the disease or other medical condition is cancer.
  • cancers are colorectal, pancreatic, thyroid, lung, acute myeloid leukemia, or glioblastoma.
  • the body fluid is blood, plasma, serum, urine, or combinations thereof.
  • the subject is a human.
  • the microvesicles isolated from a bodily fluid are enriched for those originating from a specific cell type, such as specific cell type is lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, or fetus cells.
  • a specific cell type such as specific cell type is lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, or fetus cells.
  • microvesicular surface molecules are used to enrich for microvesicles from a specific cell.
  • the microvesicular surface molecules are surface antigens associated with tumor cells, such as 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.
  • the absence of a microvesicular surface molecule is used to enrich for microvesicles from a specific cell type, such as the surface molecules CD80 or CD86.
  • the isolation of microvesicles from a specific cell type is accomplished by using antibodies, aptamers, aptamer analogs, or molecularly imprinted polymers.
  • one or more nucleic acids are extracted from the one or more microvesicles prior to analysis.
  • the nucleic acids are DNA.
  • the nucleic acids are RNA.
  • the RNA are reverse-transcribed into complementary DNA.
  • the nucleic acids are analyzed directly without an amplification step.
  • the nucleic acids are amplified prior to analysis.
  • the nucleic acid amplifications are carried out by polymerase chain reaction (PCR) and its variants such as in situ PCR, quantitative PCR, nested PCR; self-sustained sequence replication and its variants; transcriptional amplification system and its variants; Qb Replicase and its variants; or cold-PCR.
  • PCR polymerase chain reaction
  • the subject is a human colorectal cancer patient.
  • the present invention may be as defined in any one of the following numbered paragraphs.
  • FIG. 1 Illustration of a bioanalyzer profile of RNAs extracted from serum exosomes.
  • the nucleotide (nt) size is depicted on the x-axis, and the quantity is depicted on the y-axis as fluorescent units (FU).
  • FIG. 2 Amplification plot illustrating the detection of Kras Exon 4 (a region that is not commonly mutated and therefore detects both mutated and wild type KRAS) using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis, and the ⁇ Rn (normalised fluorescence emission) versus cycle is shown on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the Scorpion kit manufacturer, DxS of Manchester, England (now known as QIAGEN, Manchester, Ltd.; hereinafter referred to as “DxS”)).
  • the labels are as follows:
  • FIG. 3 Illustration of a result detecting Kras G12D mutation using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis and the ⁇ Rn (normalized fluorescence emission) versus cycle on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the kit manufacturer, DxS).
  • the labels are as follows:
  • FIG. 4( a ) Positive and negative control reaction for the detection of Kras Exon 4 (present in both mutated and wild type KRAS) using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis and the ⁇ Rn (normalized fluorescence emission) versus cycle on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the kit manufacturer, DxS).
  • the labels are as follows:
  • FIG. 4( b ) Positive and negative control reaction for the detection of the Kras G12A mutation using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis and the ⁇ Rn (normalized fluorescence emission) versus cycle on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the kit manufacturer, DxS).
  • the labels are as follows:
  • FIG. 5( a ) Illustration of a result detecting Kras Exon 4 using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis and the ⁇ Rn (normalized fluorescence emission) versus cycle on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the kit manufacturer, DxS).
  • the labels are as follows:
  • FIG. 5( b ) Illustration of a result detecting Kras Exon 4 using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis and the ⁇ Rn (normalized fluorescence emission) versus cycle on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the kit manufacturer, DxS).
  • the labels are as follows:
  • FIG. 6( a ) Illustration of a result detecting Kras G12A mutation using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis and the ⁇ Rn (normalized fluorescence emission) versus cycle on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the kit manufacturer, DxS).
  • the labels are as follows:
  • FIG. 6( b ) Illustration of a result detecting Kras G12A mutation using a modified Scorpion® mutation detection method.
  • the PCR cycle number is shown on the x-axis and the ⁇ Rn (normalized fluorescence emission) versus cycle on the y-axis.
  • the Applied Biosystems 7500 Fast qPCR machine was used for the analysis (run in standard mode, as recommended by the kit manufacturer, DxS).
  • the labels are as follows:
  • Microvesicles are shed by eukaryotic cells, or budded off of the plasma membrane, to the exterior of the cell. These membrane vesicles are heterogeneous in size with diameters ranging from about 10 nm to about 5000 nm.
  • the small microvesicles (approximately 10 to 1000 nm, and more often approximately 30 to 200 nm in diameter) that are released by exocytosis of intracellular multivesicular bodies are referred to in the art as “exosomes.”
  • the methods and compositions described herein are equally applicable to microvesicles of all sizes; preferably 30 to 800 nm; and more preferably 30 to 200 nm.
  • exosome also refers to protein complexes containing exoribonucleases which are involved in mRNA degradation and the processing of small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) and ribosomal RNAs (rRNA) (Liu, et al. 2006; van Dijk, et al. 2007).
  • snoRNAs small nucleolar RNAs
  • snRNAs small nuclear RNAs
  • rRNA ribosomal RNAs
  • nucleic acids found within microvesicles can be used as valuable biomarkers for tumor diagnosis, characterization and prognosis by providing a genetic biomarker or profile.
  • the nucleic acids within microvesicles can also be used to monitor tumor progression over time by analyzing if other mutations are acquired during tumor progression as well as if the levels of certain mutations are becoming increased or decreased over time or over a course of treatment (Skog et al., WO 2009/100029).
  • Certain aspects of the present invention are based on the finding that the ability to analyze nucleic acids from microvesicles provides a non-invasive and sensitive method for detecting genetic aberrations.
  • This ability to detect genetic aberrations provides for the ability to detect, diagnose, monitor, treat, or evaluate a disease or other medical condition, by analyzing nucleic acid content from microvesicles.
  • nucleic acids from microvesicles may be isolated and analyzed periodically as a means to detect changes in nucleic acids.
  • Such analyses can provide valuable information regarding the state of a disease or other medical condition, at the particular point in time that the microvesicles were obtained from the subject. This information may be used to assist in the therapeutic evaluation and decision-making process for a subject having a disease or other medical condition.
  • the presence or absence of one or more mutations in a particular gene may indicate the susceptibility to, presence of, or progression of a disease or other medical condition in a subject, or may indicate the likelihood that a particular therapeutic treatment will be efficacious.
  • the KRAS mutation status is predictive of response to therapy with drugs such as cetuximab and panitumumab (also known as Erbitux and Vectibix) (anti-EGFR inhibitors) in colorectal cancer
  • Certain aspects of the present invention are based on another finding that most of the extracellular RNAs in bodily fluid of a subject are contained within microvesicles and thus protected from degradation by ribonucleases. More than 90% of extracellular RNA in total serum can be recovered in microvesicles (Skog et al., WO 2009/100029).
  • the present invention relates to methods for diagnosing, prognosing, monitoring, and treating a disease or other medical condition in a subject comprising the steps of, isolating a microvesicle fraction (or obtaining a microvesicle preparation) from a bodily fluid of a subject, and analyzing one or more nucleic acids contained within the microvesicles.
  • the nucleic acids are analyzed qualitatively and/or quantitatively, and the results are compared to results expected or obtained for one or more other subjects who have or do not have the disease or other medical condition.
  • microvesicular nucleic acid content of the subject can indicate the presence or absence of a disease or other medical condition, the progression of said disease or other medical condition (e.g., changes of tumor size and tumor malignancy), the susceptibility to a disease or other medical condition, or the efficacy of a drug or other therapeutic treatment for a particular subject.
  • a reference e.g., microvesicular nucleic acid content of one or more other individuals, or prior analyses of the microvesicular nucleic content of the same individual
  • compositions, methods and techniques described herein provide the following advantages: 1) the opportunity to selectively analyze disease- or tumor-specific nucleic acids, which may be realized by isolating disease- or tumor-specific microvesicles apart from other microvesicles within the fluid sample; 2) significantly higher yield of nucleic acid species with higher sequence integrity as compared to the yield/integrity obtained by extracting nucleic acids directly from the fluid sample; 3) scalability, e.g.
  • the sensitivity can be increased by isolating more microvesicles from a larger volume of serum; 4) purer nucleic acids in that protein and lipids, debris from dead cells, and other potential contaminants and PCR inhibitors are excluded from the microvesicle preparation before the nucleic acid extraction step; and 5) more choices in nucleic acid extraction methods as microvesicle preparations are of much smaller volume than that of the starting serum, making it possible to extract nucleic acids from these microvesicle preparations using small volume column filters.
  • the microvesicles are preferably isolated from a bodily fluid from a subject.
  • a “bodily fluid” refers to a sample of fluid isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, blood, plasma, serum, urine, sputum, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof.
  • subject is intended to include all animals shown to or expected to have microvesicles.
  • the subject is a mammal, a human or nonhuman primate, a dog, a cat, a horse, a cow, other farm animals, or a rodent (e.g. mice, rats, guinea pig, etc.).
  • rodent e.g. mice, rats, guinea pig, etc.
  • subject and “individual” are used interchangeably herein.
  • Methods of isolating microvesicles from a biological sample are known in the art. For example, a method of differential centrifugation is described in a paper by Raposo, et al. (Raposo, et al. 1996), and similar methods are detailed in the Examples section herein. Methods of anion exchange and/or gel permeation chromatography are described in U.S. Pat. Nos. 6,899,863 and 6,812,023. Methods of sucrose density gradients or organelle electrophoresis are described in U.S. Pat. No. 7,198,923. A method of magnetic activated cell sorting (MACS) is described in (Taylor and Gercel-Taylor 2008).
  • MCS magnetic activated cell sorting
  • microvesicles can be identified and isolated from bodily fluid of a subject by a recently developed microchip technology that uses a unique microfluidic platform to efficiently and selectively separate tumor derived microvesicles.
  • This technology as described in a paper by Nagrath, et al. (Nagrath, et al. 2007), can be adapted to identify and separate microvesicles using similar principles of capture and separation as taught in the paper.
  • methods of isolating microvesicles from urine samples are described in a paper by Miranda, et al. (Miranda, et al. 2010) and in Russo, et al., PCT/US10/042365, filed Jul. 16, 2010 (expected to publish in 2011).
  • the microvesicles isolated from a bodily fluid are enriched for those originating from a specific cell type, for example, lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, fetus cells.
  • a specific cell type for example, lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, fetus cells.
  • surface molecules may be used to identify, isolate and/or enrich for microvesicles from a specific donor cell type (Al-Nedawi, et al. 2008; Taylor and Gercel-Taylor 2008).
  • microvesicles originating from distinct cell populations can be analyzed for their nucleic acid content.
  • tumor (malignant and non-malignant) microvesicles carry tumor-associated surface antigens and may be detected, isolated and/or enriched via these specific tumor-associated surface antigens.
  • the surface antigen is epithelial-cell-adhesion-molecule (EpCAM), which is specific to microvesicles from carcinomas of lung, colorectal, breast, prostate, head and neck, and hepatic origin, but not of hematological cell origin (Balzar, et al. 1999; Went, et al. 2004).
  • the surface antigen is CD24, which is a glycoprotein specific to urine microvesicles (Keller, et al. 2007).
  • the surface antigen is selected from a group of molecules CD70, carcinoembryonic antigen (CEA), EGFR, EGFRvIII and other variants, Fas ligand, TRAIL, tranferrin receptor, p38.5, p97 and HSP72.
  • tumor specific microvesicles may be characterized by the lack of surface markers, such as CD80 and CD86.
  • the isolation of microvesicles from specific cell types can be accomplished, for example, by using antibodies, aptamers, aptamer analogs or molecularly imprinted polymers specific for a desired surface antigen.
  • the surface antigen is specific for a cancer type.
  • the surface antigen is specific for a cell type which is not necessarily cancerous.
  • U.S. Pat. No. 7,198,923. As described in, e.g., U.S. Pat. Nos. 5,840,867 and 5,582,981, WO 2003/050290 and a publication by Johnson, et al. (Johnson, et al.
  • aptamers and their analogs specifically bind surface molecules and can be used as a separation tool for retrieving cell type-specific microvesicles.
  • Molecularly imprinted polymers also specifically recognize surface molecules as described in, e.g., U.S. Pat. Nos. 6,525,154, 7,332,553 and 7,384,589 and a publication by Bossi, et al. (Bossi, et al. 2007) and are a tool for retrieving and isolating cell type-specific microvesicles.
  • Bossi, et al. Bossi, et al. 2007
  • Nucleic acid molecules can be isolated from a microvesicle using any number of procedures, which are well-known in the art, the particular extraction procedure chosen being appropriate for the particular biological sample. For example, methods for extracting nucleic acids from urinary microvesicles are described in Miranda, et al. (Miranda, et al. 2010) and in Russo, et al., PCT/US10/042365, filed Jul. 16, 2010 (expected to publish in 2011), each of which is incorporated herein for its teaching of these methods. In some instances, with some techniques, it may also be possible to analyze the nucleic acid without extraction from the microvesicle.
  • the extracted nucleic acids are analyzed directly without an amplification step.
  • Direct analysis may be performed with different methods including, but not limited to, nanostring technology.
  • NanoString technology enables identification and quantification of individual target molecules in a biological sample by attaching a color coded fluorescent reporter to each target molecule. This approach is similar to the concept of measuring inventory by scanning barcodes. Reporters can be made with hundreds or even thousands of different codes allowing for highly multiplexed analysis. The technology is described in a publication by Geiss, et al. (Geiss, et al. 2008) and is incorporated herein by reference for this teaching.
  • nucleic acid of the microvesicle it may be beneficial or otherwise desirable to amplify the nucleic acid of the microvesicle prior to analyzing it.
  • Methods of nucleic acid amplification are commonly used and generally known in the art, many examples of which are described herein. If desired, the amplification can be performed such that it is quantitative. Quantitative amplification will allow quantitative determination of relative amounts of the various nucleic acids, to generate a profile as described below.
  • the extracted nucleic acid is DNA.
  • the extracted nucleic acid is RNA.
  • RNAs are preferably reverse-transcribed into complementary DNAs. Such reverse transcription may be performed alone or in combination with an amplification step.
  • a method combining reverse transcription and amplification steps is reverse transcription polymerase chain reaction (RT-PCR), which may be further modified to be quantitative, e.g., quantitative RT-PCR as described in U.S. Pat. No. 5,639,606, which is incorporated herein by reference for this teaching.
  • RT-PCR reverse transcription polymerase chain reaction
  • Nucleic acid amplification methods include, without limitation, polymerase chain reaction (PCR) (U.S. Pat. No. 5,219,727) and its variants such as in situ polymerase chain reaction (U.S. Pat. No. 5,538,871), quantitative polymerase chain reaction (U.S. Pat. No. 5,219,727), nested polymerase chain reaction (U.S. Pat. No. 5,556,773), self sustained sequence replication and its variants (Guatelli, et al. 1990), transcriptional amplification system and its variants (Kwoh, et al. 1989), Qb Replicase and its variants (Miele, et al. 1983), cold-PCR (Li, et al.
  • PCR polymerase chain reaction
  • U.S. Pat. No. 5,219,727 in situ polymerase chain reaction
  • quantitative polymerase chain reaction U.S. Pat. No. 5,219,727
  • nested polymerase chain reaction U.S. Pat
  • nucleic acids present in the microvesicles is quantitative and/or qualitative.
  • amounts (expression levels), either relative or absolute, of specific nucleic acids of interest within the microvesicles are measured with methods known in the art.
  • species of specific nucleic acids of interest within the microvesicles, whether wild type or variants, are identified with methods known in the art.
  • Genetic aberrations is used herein to refer to the nucleic acid amounts as well as nucleic acid variants within the microvesicles.
  • genetic aberrations include, without limitation, over-expression of a gene (e.g., oncogenes) or a panel of genes, under-expression of a gene (e.g., tumor suppressor genes such as p53 or RB) or a panel of genes, alternative production of splice variants of a gene or a panel of genes, gene copy number variants (CNV) (e.g.
  • CNV gene copy number variants
  • DNA double minutes DNA double minutes
  • nucleic acid modifications e.g., methylation, acetylation and phosphorylations
  • single nucleotide polymorphisms SNPs
  • chromosomal rearrangements e.g., inversions, deletions and duplications
  • mutations insertions, deletions, duplications, missense, nonsense, synonymous or any other nucleotide changes
  • nucleic acid modifications can be assayed by methods described in, e.g., U.S. Pat. No.
  • methylation profiles may be determined by Illumina DNA Methylation OMA003 Cancer Panel.
  • SNPs and mutations can be detected by hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatched heteroduplex (Cotton, et al. 1988), ribonuclease cleavage of mismatched bases (Myers, et al. 1985), mass spectrometry (U.S. Pat. Nos. 6,994,960, 7,074,563, and 7,198,893), nucleic acid sequencing, single strand conformation polymorphism (SSCP) (Orita, et al.
  • SSCP single strand conformation polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • TGGE temperature gradient gel electrophoresis
  • RFLP restriction fragment length polymorphisms
  • OPA oligonucleotide ligation assay
  • ASPCR allele-specific PCR
  • LCR allele-specific PCR
  • nucleic acid variants e.g., DNA or RNA modifications, single nucleotide polymorphisms (SNPs) and mutations (e.g., missense, nonsense, insertions, deletions, duplications) may also be analyzed within microvesicles from bodily fluid of a subject, including pregnant females where microvesicles derived from the fetus may be in serum as well as amniotic fluid.
  • SNPs single nucleotide polymorphisms
  • mutations e.g., missense, nonsense, insertions, deletions, duplications
  • the RAS family of oncogenes is commonly mutated in cancer.
  • the Ras family consists of Hras, Kras, and Nras genes, all of which encode for GTP-binding proteins that act to transmit signals from receptor tyrosine kinases to downstream modulators of cell growth.
  • an activating mutation in a Ras gene is found.
  • the mutated Ras gene is the Kras gene.
  • Kras mutations are found in roughly 50% of all colorectal cancers (Jancik et al. 2010).
  • Ras has been shown to play an important role in the expression of matrix metalloproteinases, as well as other processes that promote tumor invasion and metastasis.
  • the ability to detect genetic aberrations in microvesicles of a subject provides a useful model for practicing companion diagnostics. Based on analyses of nucleic acids from the microvesicles of a subject having a disease or other medical condition, therapeutic treatment may be tailored for that subject. For example, a companion diagnostic test kit may be developed to test for one or more mutations in the Kras gene. Based on the presence or absence of such mutations, a particular therapeutic treatment may or may not be recommended.
  • the Kras protein regulates two signaling pathways: (1) PI 3-kinase/phosphatase and tensin homolog (PTEN)/AKT; and (2) RAF/MEK/ERK. These pathways are popular targets for anti-cancer therapies, including drugs which target Epidermal Growth Factor Receptor (EGFR), upstream from Kras. When bound to its ligand, EGFR initiates tyrosine kinase activity, activating Kras, and the signaling pathways (Quest Diagnostics, KRAS Mutation Analysis, Reference Materials (taken from website at http://www.questdiagnostics.com/hcp/intguide/jsp/showintguidepage.jsp?fn TS_KRAS.htm) (last visited Sep. 9, 2010).
  • EGFR Epidermal Growth Factor Receptor
  • Existing therapies that target EGFR are used to treat various cancers, including colorectal cancer and non-small-cell cancer. These therapies employ either: (a) monoclonal antibodies, such as cetuximab or panitumumab, that abrogate ligand binding and, thus, EGFR activation; or (b) tyrosine kinease inhibitors, such as erlotinib, that prevent activation of the signaling pathways.
  • monoclonal antibodies such as cetuximab or panitumumab
  • tyrosine kinease inhibitors such as erlotinib
  • Detection of one or more nucleotide variants can be accomplished by performing a nucleotide variant screen on the nucleic acids within the microvesicles.
  • a nucleotide variant screen can be as wide or narrow as determined necessary or desirable by the skilled practitioner. It can be a wide screen (set up to detect all possible nucleotide variants in genes known to be associated with one or more cancers or disease states). Where one specific cancer or disease is suspected or known to exist, the screen can be specific to that cancer or disease.
  • a brain tumor/brain cancer screen e.g., set up to detect all possible nucleotide variants in genes associated with various clinically distinct subtypes of brain cancer or known drug-resistant or drug-sensitive mutations of that cancer).
  • the analysis is of a profile of the amounts (levels) of specific nucleic acids present in the microvesicle, herein referred to as a “quantitative nucleic acid profile” of the microvesicles.
  • the analysis is of a profile of the species of specific nucleic acids present in the microvesicles (both wild type as well as variants), herein referred to as a “nucleic acid species profile.”
  • a term used herein to refer to a combination of these types of profiles is “genetic profile” which refers to the determination of the presence or absence of nucleotide species, variants and also increases or decreases in nucleic acid levels.
  • a profile can be a genome wide profile (set up to detect all possible expressed genes or DNA sequences). It can be narrower as well, such as a cancer wide profile (set up to detect all possible genes or nucleic acids derived therefrom, or known to be associated with one or more cancers). Where one specific cancer is suspected or known to exist, the profile can be specific to that cancer (e.g., set up to detect all possible genes or nucleic acids derived therefrom, associated with various clinically distinct subtypes of that cancer or known drug-resistant or sensitive mutations of that cancer).
  • nucleic acids are to be amplified and/or analyzed can be selected by the skilled practitioner.
  • the entire nucleic acid content of the exosomes or only a subset of specific nucleic acids which are likely or suspected of being influenced by the presence of a disease or other medical condition such as cancer, can be amplified and/or analyzed.
  • the identification of a nucleic acid aberration(s) in the analyzed microvesicle nucleic acid can be used to diagnose the subject for the presence of a disease such as cancer, hereditary diseases or viral infection with which that aberration(s) is associated.
  • analysis for the presence or absence of one or more nucleic acid variants of a gene specific to a cancer e.g. the Kras mutation
  • mutations of a gene which is associated with a disease such as cancer are detected by analysis of nucleic acids in microvesicles, which nucleic acids are derived from the genome itself in the cell of origin or exogenous genes introduced through viruses.
  • the nucleic acid sequences may be complete or partial, as both are expected to yield useful information in diagnosis and prognosis of a disease.
  • the sequences may be sense or anti-sense to the actual gene or transcribed sequences. The skilled practitioner will be able to devise detection methods for a nucleotide variance from either the sense or anti-sense nucleic acids which may be present in a microvesicle.
  • probes which are specific for the nucleotide sequences which directly flank, or contain the nucleotide variances.
  • probes can be designed by the skilled practitioner given the knowledge of the gene sequences and the location of the nucleic acid variants within the gene.
  • probes can be used to isolate, amplify, and/or actually hybridize to detect the nucleic acid variants, as described in the art and herein.
  • Determining the presence or absence of a particular nucleotide variant or plurality of variants in the nucleic acid within microvesicles from a subject can be performed in a variety of ways. A variety of methods are available for such analysis, including, but not limited to, PCR, hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatches, mass spectrometry or DNA sequencing, including minisequencing.
  • hybridization with allele specific probes can be conducted in two formats: 1) allele specific oligonucleotides bound to a solid phase (glass, silicon, nylon membranes) and the labeled sample in solution, as in many DNA chip applications, or 2) bound sample (often cloned DNA or PCR amplified DNA) and labeled oligonucleotides in solution (either allele specific or short so as to allow sequencing by hybridization). Diagnostic tests may involve a panel of variances, often on a solid support, which enables the simultaneous determination of more than one variance.
  • determining the presence of at least one nucleic acid variance in the microvesicle nucleic acid entails a haplotyping test. Methods of determining haplotypes are known to those of skill in the art, as for example, in WO 00/04194.
  • the determination of the presence or absence of a nucleic acid variant(s) involves determining the sequence of the variant site or sites (the exact location within the sequence where the nucleic acid variation from the norm occurs) by methods such as polymerase chain reaction (PCR), chain terminating DNA sequencing (U.S. Pat. No. 5547859), minisequencing (Fiorentino, et al. 2003), oligonucleotide hybridization, pyrosequencing, Illumina genome analyzer, deep sequencing, mass spectrometry or other nucleic acid sequence detection methods.
  • PCR polymerase chain reaction
  • minisequencing Fiorentino, et al. 2003
  • oligonucleotide hybridization pyrosequencing
  • Illumina genome analyzer Illumina genome analyzer
  • deep sequencing deep sequencing
  • mass spectrometry or other nucleic acid sequence detection methods.
  • the diagnostic test comprises amplifying a segment of DNA or RNA (generally after converting the RNA to complementary DNA) spanning one or more known variants in the desired gene sequence. This amplified segment is then sequenced and/or subjected to electrophoresis in order to identify nucleotide variants in the amplified segment.
  • the invention provides a method of screening for nucleotide variants in the nucleic acid of microvesicles isolated as described herein. This can be achieved, for example, by PCR or, alternatively, in a ligation chain reaction (LCR) (Landegren, et al. 1988; Nakazawa, et al. 1994; Abravaya, et al. 1995). LCR can be particularly useful for detecting point mutations in a gene of interest (Abravaya, et al. 1995).
  • LCR ligation chain reaction
  • the LCR method comprises the steps of designing degenerate primers for amplifying the target sequence, the primers corresponding to one or more conserved regions of the nucleic acid corresponding to the gene of interest, amplifying PCR products with the primers using, as a template, a nucleic acid obtained from a microvesicle, and analyzing the PCR products. Comparison of the PCR products of the microvesicle nucleic acid to a control sample (either having the nucleotide variant or not) indicates variants in the microvesicle nucleic acid. The change can be either an absence or presence of a nucleotide variant in the microvesicle nucleic acid, depending upon the control.
  • the invention provides a method of screening for nucleotide variants of the Kras gene isolated from microvesicles.
  • the detection of mutations in the Kras gene is performed by a real-time PCR assay using a KRAS PCR Kit (Qiagen®). While the KRAS PCR Kit is intended to be used with genomic DNA samples, it may also be employed for use with other nucleic acid samples that are not derived from genomic DNA, provided the KRAS PCR Kit is modified. For example, in another embodiment, the KRAS PCR Kit is modified to quantitatively detect Kras mutants from RNA samples. Isolated RNA is reverse transcribed via Reverse Transcriptase (RT) into complementary DNA (cDNA).
  • RT Reverse Transcriptase
  • the cDNA sample is purified using standard techniques known in the art for purifying DNA, including, e.g., by ethanol precipitation and via purification columns. Alternatively, the cDNA may be diluted. Once it is substantially free from the impurities of the RT reaction, either by purification or dilution, the cDNA sample is subject to the amplification steps of the KRAS PCR Kit. Because of this modification, i.e., the added step of purifying the cDNA template prior to amplification, the KRAS PCR Kit may be employed to detect, quantify, and analyze RNA samples.
  • microvesicles Many methods of diagnosis performed on a tumor biopsy sample can be performed with microvesicles since tumor cells are known to shed microvesicles into bodily fluid and the genetic aberrations within these microvesicles reflect those within tumor cells as demonstrated herein. Furthermore, methods of diagnosis using microvesicles have characteristics that are absent in methods of diagnosis performed directly on a tumor biopsy sample.
  • one particular advantage of the analysis of microvesicular nucleic acids, as opposed to other forms of sampling of tumor/cancer nucleic acid is the availability for analysis of tumor/cancer nucleic acids derived from all foci of a tumor or genetically heterogeneous tumors present in an individual. Biopsy samples are limited in that they provide information only about the specific focus of the tumor from which the biopsy is obtained.
  • the microvesicle fraction from a bodily fluid of a subject is pre-treated with DNase to eliminate or substantially eliminate all of any DNA located on the surface of the microvesicles or outside of the microvesicles.
  • DNase DNAse pre-treatment
  • short DNA fragments on the outside of microvesicles may remain and co-isolate with nucleic acids extracted from inside the microvesicles.
  • elimination of all or substantially all of any DNA associated with the outside or surface of microvesicles by pre-treatment of the microvesicles with DNase has the ability to enrich for nucleic acid from within the microvesicles.
  • Identification of genetic aberrations associated with specific diseases and/or medical conditions by the methods described herein can also be used for prognosis and treatment decisions of an individual diagnosed with a disease or other medical condition such as cancer. Identification of the genetic basis of a disease and/or medical condition provides useful information guiding the treatment of the disease and/or medical condition. For example, many forms of chemotherapy have been shown to be more effective on cancers with specific genetic abnormalities/aberrations. One example is the effectiveness of EGFR-targeting treatments with medicines, such as the kinase inhibitors gefitinib and erlotinib.
  • Such treatments have been shown to be more effective on cancer cells whose EGFR gene harbors specific nucleotide mutations in the kinase domain of the EGFR protein (U.S. Patent publication 20060147959).
  • the presence of at least one of the identified nucleotide variants in the kinase domain of EGFR nucleic acid message indicates that a patient will likely benefit from treatment with the EGFR-targeting compound gefitinib or erlotinib.
  • Such nucleotide variants can be identified in nucleic acids present in microvesicles by the methods described herein.
  • Other aspects of the present invention relate to a method for monitoring disease (e.g. cancer) progression in a subject, and also to a method for monitoring disease recurrence in an individual.
  • These methods comprise the steps of isolating microvesicles from a bodily fluid of an individual, as discussed herein, and analyzing nucleic acid within the microvesicles as discussed herein (e.g. to create a genetic profile of the microvesicles).
  • the presence/absence of a certain genetic aberration/profile is used to indicate the presence/absence of the disease (e.g. cancer) in the subject as discussed herein.
  • the process is performed periodically over time, and the results reviewed, to monitor the progression or regression of the disease, or to determine recurrence of the disease.
  • a change in the genetic profile indicates a change in the disease state in the subject.
  • the period of time to elapse between sampling of microvesicles from the subject, for performance of the isolation and analysis of the microvesicle will depend upon the circumstances of the subject, and is to be determined by the skilled practitioner.
  • Such a method would prove extremely beneficial when analyzing a nucleic acid from a gene that is associated with the therapy undergone by the subject.
  • a gene which is targeted by the therapy can be monitored for the development of mutations which make it resistant to the therapy, upon which time the therapy can be modified accordingly.
  • the monitored gene may also be one which indicates specific responsiveness to a specific therapy.
  • aspects of the present invention also relate to the fact that a variety of non-cancer diseases and/or medical conditions also have genetic links and/or causes, and such diseases and/or medical conditions can likewise be diagnosed and/or monitored by the methods described herein.
  • Many such diseases are metabolic, infectious or degenerative in nature.
  • diabetes e.g. diabetes insipidus
  • V2R vasopressin type 2 receptor
  • kidney fibrosis in which the genetic profiles for the genes of collagens, fibronectin and TGF- ⁇ are changed. Changes in the genetic profile due to substance abuse (e.g. a steroid or drug use), viral and/or bacterial infection, and hereditary disease states can likewise be detected by the methods described herein.
  • nephropathy diabetes insipidus, diabetes mellitus, diabetes type I, diabetes II, renal disease glomerulonephritis, bacterial or viral glomerulonephritides, IgA nephropathy, Henoch-Schonlein Purpura, membranoproliferative glomerulonephritis, membranous nephropathy, Sjogren's syndrome, nephrotic syndrome minimal change disease, focal glomerulosclerosis and related disorders, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, GU tract inflammatory disease, Pre-clampsia, renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis, genetic renal disease, medullary cystic, medullar sponge, polycystic kidney disease, autosomal dominant polycys
  • Selection of an individual from whom the microvesicles are isolated is performed by the skilled practitioner based upon analysis of one or more of a variety of factors. Such factors for consideration are whether the subject has a family history of a specific disease (e.g. a cancer), has a genetic predisposition for such a disease, has an increased risk for such a disease due to family history, genetic predisposition, other disease or physical symptoms which indicate a predisposition, or environmental reasons. Environmental reasons include lifestyle, exposure to agents which cause or contribute to the disease such as in the air, land, water or diet. In addition, having previously had the disease, being currently diagnosed with the disease prior to therapy or after therapy, being currently treated for the disease (undergoing therapy), being in remission or recovery from the disease, are other reasons to select an individual for performing the methods.
  • a specific disease e.g. a cancer
  • genetic predisposition for such a disease
  • environmental reasons include lifestyle, exposure to agents which cause or contribute to the disease such as in the air, land, water or diet.
  • the methods described herein are optionally performed with the additional step of selecting a gene or nucleic acid for analysis, prior to the analysis step. This selection can be based on any predispositions of the subject, or any previous exposures or diagnosis, or therapeutic treatments experienced or concurrently undergone by the subject.
  • the cancer diagnosed, monitored or otherwise profiled can be any kind of cancer.
  • the methods and compositions of the present invention are equally applicable to detection, diagnosis and prognosis of non-malignant tumors in an individual (e.g. neurofibromas, meningiomas and schwannomas).
  • microvesicles 0.6-2 milliliter serum was filtered through a 0.8 ⁇ m filter to remove any cell contamination. Microvesicles were then pelleted by ultracentrifugation at 110,000 ⁇ g for 70 minutes.
  • RNAse inhibitor solution for the extraction of RNA from microvesicles, the pelleted microvesicles were incubated in an RNAse inhibitor solution for 20 minutes at room temperature.
  • the RNase inhibitor can be obtained from various known vendors, e.g., SUPERase-In (Ambion Inc).
  • Total RNA was then extracted from the RNAse-treated microvesicles using miRNeasy RNA extraction kit (Qiagen).
  • miRNeasy RNA extraction kit Qiagen
  • various commercial RNA extraction kits such as the QIAamp RNA Blood Mini Kit from Qiagen, QIAamp viral RNA mini kit (Qiagen) and the MirVana RNA isolation kit from Ambion Inc. may be used according to the manufacturer's protocols. After treatment with DNAse according to the manufacturer's protocol, total RNA was eluted in 30 ⁇ l nuclease-free water.
  • the extracted RNAs were then analyzed using a Bioanalyzer RNA chip (Agilent Technologies) to confirm the quality of the RNA. See FIG. 1 .
  • the isolated RNAs were then analyzed by a quantitative PCR assay; namely, a modified Scorpion® Kras mutation detection method.
  • the off-the-shelf KRAS Mutation Test Kit is intended for the detection of 7 somatic mutations in the KRAS oncogene. The kit is marketed and sold for use on DNA samples and will provide a qualitative assessment of mutation status.
  • the isolated RNAs were first reverse-transcribed into cDNAs using a standard reverse transcription method, e.g., the Sensiscript RT kit (Qiagen).
  • RNA-reverse-transcribed cDNAs were then purified using micro-columns with filters capable of retaining molecules above 30 kDa or 20-40 nucleotides.
  • the purified cDNAs were used in the Scorpion® Kras mutation detection PCR reactions.
  • Kras Exon 4 can be readily detected when the cDNAs from serum and plasma exosomes were used.
  • the number of cycles for the serum exosomes (C) was less than the number of cycles for the plasma exosomes (D), suggesting that serum RNA was more abundant than plasma RNA.
  • the positive controls can be detected and the negative controls cannot.
  • the Kras G12D mutation can be readily detected when the cDNAs from serum and plasma exosomes were used.
  • the number of cycles for the serum exosomes (C) was similar to the number of cycles for the plasma exosomes (D), suggesting that serum exosomes RNA was as abundant as plasma exosome RNA.
  • the positive controls can be detected and the negative controls cannot.
  • Scorpion® Kras mutation detection PCR reactions to detect Kras Exon 4 were performed on positive and negative controls ( FIG. 4( a )).
  • Scorpion® Kras mutation detection PCR reactions to detect Kras G12A mutations were performed on positive and negative controls ( FIG. 4( b )). As is shown in FIG. 4 , the positive controls can be detected and the negative controls cannot.
  • serum and plasma samples were obtained for the following analysis, from a patient diagnosed with colorectal cancer and having a Kras G12A mutation, confirmed by pathology evaluation of a biopsy.
  • Microvesicles were isolated as described previously in Example 1. Prior to isolation of nucleic acids from the microvesicles, a subset of the isolated microvesicles was pre-treated with DNase (TurboTM DNase (Ambion®)) in order to eliminate or substantially eliminate any DNA located on the surface of the microvesicles or outside of the microvesicles. The remaining subset of microvesicles was left untreated. Samples of nucleic acid (both RNA and DNA) were obtained for analysis as described previously in Example 1. The RNA was reverse-transcribed into cDNA, as described in Example 1. The purified cDNA ( FIG. 5( a )) and DNA ( FIG.
  • RNA and DNA containing Kras Exon 4 were more abundant in the samples that came from microvesicles that were optionally pre-treated with DNase, than those that came from untreated microvesicles. These findings suggest that the DNase pre-treatment may have the effect of eliminating DNA associated with the microvesicles that might otherwise contaminate the subsequent PCR reaction, and thereby enrich for mutant nucleic acids.
  • DNase pre-treatment in method of analyzing the Kras G12A mutation in RNA and DNA using microvesicles isolated from serum and plasma samples, treated with DNase.
  • serum and plasma samples were obtained for the following analysis, from the same patient as in Example 3, diagnosed with colorectal cancer and having a Kras G12A mutation, confirmed by pathology evaluation of a biopsy.
  • the purified cDNA ( FIG. 6( a )) and DNA ( FIG. 6( b )) were used in Scorpion® Kras mutation detection PCR reactions to detect Kras G12A mutations. As can be seen in FIGS.
  • the Scorpion® Kras mutation detection PCR reactions were able to successfully detect the Kras G12A mutant in DNA obtained directly from microvesicles, as well as from RNA-reverse-transcribed cDNA, whether or not the microvesicles were pre-treated with DNase. As can also be seen in FIGS.
  • RNA and DNA containing the Kras G12A mutation were more abundant in the samples that came from microvesicles that were pre-treated with DNase, than those that came from untreated microvesicles, supporting the suggestion that DNase pre-treatment has the potential to eliminate DNA associated with the microvesicles that might otherwise contaminate the subsequent PCR reaction, and thereby enrich for mutant nucleic acids.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014201092A1 (en) * 2013-06-11 2014-12-18 Dana-Farber Cancer Institute, Inc. Non-invasive blood based monitoring of genomic alterations in cancer
US20150218651A1 (en) * 2012-08-17 2015-08-06 Cornell University Use of dna in circulating exosomes as a diagnostic marker for metastatic disease
WO2016077709A1 (en) * 2014-11-14 2016-05-19 Liquid Genomics, Inc. Use of circulating cell-free rna for diagnosis and/or monitoring cancer
US20160237422A1 (en) * 2011-05-11 2016-08-18 Exosome Diagnostics, Inc. Nucleic acid extraction from heterogeneous biological materials
US9939443B2 (en) 2012-12-19 2018-04-10 Caris Life Sciences Switzerland Holdings Gmbh Compositions and methods for aptamer screening
US9958448B2 (en) 2012-10-23 2018-05-01 Caris Life Sciences Switzerland Holdings Gmbh Aptamers and uses thereof
US10844436B2 (en) 2014-04-01 2020-11-24 Cornell University Use of double-stranded DNA in exosomes: a novel biomarker in cancer detection
US10942184B2 (en) 2012-10-23 2021-03-09 Caris Science, Inc. Aptamers and uses thereof
US11397182B2 (en) 2014-10-07 2022-07-26 Cornell University Methods for prognosing and preventing metastatic liver disease
US11899024B2 (en) 2017-07-12 2024-02-13 Exosome Diagnostics, Inc. Treatment and diagnosis of parkinson's disease using isolated and enriched populations of biofluid-derived extracellular vesicles

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3461912B1 (de) 2009-09-09 2022-07-13 The General Hospital Corporation Verwendung von mikrovesikeln bei der analyse von nukleinsäureprofilen
US20130029339A1 (en) 2009-09-09 2013-01-31 The General Hospital Corporation Use of microvesicles in analyzing kras mutations
WO2012031008A2 (en) 2010-08-31 2012-03-08 The General Hospital Corporation Cancer-related biological materials in microvesicles
WO2012064993A1 (en) 2010-11-10 2012-05-18 Exosome Diagnosties, Inc. Method for isolation of nucleic acid containing particles and extraction of nucleic acids therefrom
CA2844671A1 (en) * 2011-08-08 2013-02-14 Caris Life Sciences Luxembourg Holdings, S.A.R.L. Biomarker compositions and methods
EP2748335B1 (de) 2011-08-22 2018-10-03 Exosome Diagnostics, Inc. Urinbiomarker
WO2013071239A1 (en) 2011-11-10 2013-05-16 Exosome Diagnostics, Inc. Cerebrospinal fluid assay
EP2780473B1 (de) 2011-11-17 2017-07-19 The Johns Hopkins University Differenzialidentifizierung von zysten
WO2013134786A2 (en) * 2012-03-09 2013-09-12 Caris Life Sciences Luxembourg Holdings, S.A.R.L. Biomarker compositions and methods
EP2903597B1 (de) * 2012-10-03 2019-12-18 Exosome Diagnostics Inc. Verwendung von mikrovesikeln bei der diagnose, prognose und behandlung von krankheiten und medizinischen leiden
JP6297676B2 (ja) 2013-05-06 2018-03-20 日立化成株式会社 標的分子を捕捉するためのデバイス及び方法
JP2016526922A (ja) 2013-08-06 2016-09-08 エクソサム ダイアグノスティクス，インコーポレイティド 尿バイオマーカーコホート、遺伝子発現特性、およびその使用の方法
WO2015144184A1 (en) * 2014-03-26 2015-10-01 University Of Copenhagen Use of timp-1 as a biomarker in the egf-receptor inhibitor treatment of metastatic colorectal cancer
US10266895B2 (en) 2014-11-05 2019-04-23 Hitachi Chemical Company Ltd. Exosomes and microvesicles in intestinal luminal fluids and stool and use of same for the assessment of inflammatory bowel disease
JP6631852B2 (ja) * 2014-11-12 2020-01-15 日立化成株式会社 臓器障害を診断するための方法および装置
US11028443B2 (en) 2015-08-31 2021-06-08 Showa Denko Materials Co., Ltd. Molecular methods for assessing urothelial disease
US11702702B2 (en) 2016-04-15 2023-07-18 Predicine, Inc. Systems and methods for detecting genetic alterations
US11174503B2 (en) 2016-09-21 2021-11-16 Predicine, Inc. Systems and methods for combined detection of genetic alterations

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100184046A1 (en) * 2008-11-12 2010-07-22 Caris Mpi, Inc. Methods and systems of using exosomes for determining phenotypes
US20110081651A1 (en) * 2005-06-28 2011-04-07 Genentech, Inc. Egfr and kras mutations

Family Cites Families (92)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5639611A (en) 1988-12-12 1997-06-17 City Of Hope Allele specific polymerase chain reaction
US5219727A (en) 1989-08-21 1993-06-15 Hoffmann-Laroche Inc. Quantitation of nucleic acids using the polymerase chain reaction
US5840867A (en) 1991-02-21 1998-11-24 Gilead Sciences, Inc. Aptamer analogs specific for biomolecules
CA2074214C (en) 1991-07-23 2000-02-22 Will Bloch Improvements in the in situ pcr
US5582981A (en) 1991-08-14 1996-12-10 Gilead Sciences, Inc. Method for identifying an oligonucleotide aptamer specific for a target
US5605798A (en) 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
GB9306053D0 (en) 1993-03-24 1993-05-12 Nycomed Pharma As Method and assay
US5639606A (en) 1993-04-06 1997-06-17 The University Of Rochester Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction
US5547859A (en) 1993-08-02 1996-08-20 Goodman; Myron F. Chain-terminating nucleotides for DNA sequencing methods
US5556773A (en) 1993-08-06 1996-09-17 Yourno; Joseph Method and apparatus for nested polymerase chain reaction (PCR) with single closed reaction tubes
US6607898B1 (en) 1996-03-26 2003-08-19 Oncomedx, Inc. Method for detection of hTR and hTERT telomerase-associated RNA in plasma or serum
US6794135B1 (en) 1996-03-26 2004-09-21 Oncomedx, Inc. Method for detection of 5T4 RNA in plasma or serum
US6759217B2 (en) 1996-03-26 2004-07-06 Oncomedx, Inc. Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer
ES2347854T5 (es) 1996-03-26 2014-07-24 Michael S. Kopreski Método que permite el uso de RNA extracelular extraído de plasma o suero para detectar, monitorizar o evaluar un cáncer
EP1164203B1 (de) 1996-11-06 2007-10-10 Sequenom, Inc. DNA-Diagnostik mittels Massenspektrometrie
CA2292039A1 (en) 1997-05-28 1998-12-03 The Walter And Eliza Hall Institute Of Medical Research Nucleic acid diagnostics based on mass spectrometry or mass separation and base specific cleavage
US6004755A (en) 1998-04-07 1999-12-21 Incyte Pharmaceuticals, Inc. Quantitative microarray hybridizaton assays
WO2000004194A1 (en) 1998-07-20 2000-01-27 Variagenics, Inc. Gene sequence variances with utility in determining the treatment of disease
US6204375B1 (en) 1998-07-31 2001-03-20 Ambion, Inc. Methods and reagents for preserving RNA in cell and tissue samples
FR2788780B1 (fr) 1999-01-27 2001-03-30 Ap Cells Inc Procede de preparation de vesicules membranaires
GB9927320D0 (en) 1999-11-18 2000-01-12 Chiron Spa Exosome separation
DE60123575D1 (de) 2000-01-28 2009-10-01 Mip Technologies Ab Funktionelle monomere zur molekularen erkennung und katalyse
US20030044890A1 (en) 2000-01-31 2003-03-06 Rosen Craig A. Nucleic acids, proteins, and antibodies
US6812023B1 (en) 2000-04-27 2004-11-02 Anosys, Inc. Methods of producing membrane vesicles
US20040241176A1 (en) 2000-04-27 2004-12-02 Ap Cells. Inc. Method of producing membrane vesicles
US6913879B1 (en) 2000-07-10 2005-07-05 Telechem International Inc. Microarray method of genotyping multiple samples at multiple LOCI
US6525154B1 (en) 2000-07-20 2003-02-25 The Regents Of The University Of California Molecular imprinting for the recognition of peptides in aqueous solution
US6794447B1 (en) 2000-07-28 2004-09-21 Taylor Made Golf Co., Inc. Golf balls incorporating nanocomposite materials
CA2430379A1 (en) 2000-12-07 2002-06-13 Chiron Corporation Endogenous retroviruses up-regulated in prostate cancer
CA2414782C (en) 2001-05-11 2012-10-09 Regents Of The University Of Minnesota Intron associated with myotonic dystrophy type 2 and methods of use
US20040181049A1 (en) 2001-06-05 2004-09-16 Orr Michael S. Rna purification methods
US6696271B2 (en) 2001-08-23 2004-02-24 The Regents Of The University Of California Frozen tissue microarray technology for analysis of RNA, DNA, and proteins
WO2003023065A1 (en) 2001-09-06 2003-03-20 Syngenta Participations Ag Dna methylation patterns
US7671010B2 (en) 2002-08-30 2010-03-02 The Board Of Regents Of The University Of Texas System Compositions and methods of use of targeting peptides for diagnosis and therapy of human cancer
US20030162190A1 (en) 2001-11-15 2003-08-28 Gorenstein David G. Phosphoromonothioate and phosphorodithioate oligonucleotide aptamer chip for functional proteomics
CN1639323A (zh) 2002-03-14 2005-07-13 阿诺塞斯公司 T细胞来源囊泡的功能化及其在制备免疫原性药用组合物中的应用
US20050250100A1 (en) 2002-06-12 2005-11-10 Yoshihide Hayashizaki Method of utilizing the 5'end of transcribed nucleic acid regions for cloning and analysis
JP4384603B2 (ja) 2002-06-26 2009-12-16 コールド スプリング ハーバー ラボラトリー メチル化プロファイルを測定するための方法および組成物
CA2497288A1 (en) 2002-09-02 2004-03-11 Pamgene B.V. Novel integrated microarray analysis
US7141371B2 (en) 2002-09-06 2006-11-28 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon Methods for detecting and localizing DNA mutations by microarray
WO2004069174A2 (en) 2003-01-31 2004-08-19 Slil Biomedical Corporation Monitoring and treatment of amyotrophic lateral sclerosis
WO2005020784A2 (en) 2003-05-23 2005-03-10 Mount Sinai School Of Medicine Of New York University Surrogate cell gene expression signatures for evaluating the physical state of a subject
WO2004108770A1 (en) 2003-05-30 2004-12-16 Rensselaer Polytechnic Institute Low odor chain transfer agents for controlled radical polymerization
EP2327796A1 (de) 2003-06-10 2011-06-01 The Trustees Of Boston University Nachweismethoden für Lungenerkrankungen
EP1498144A1 (de) 2003-07-15 2005-01-19 Universite Pierre Et Marie Curie Paris Vi Extrazelluläre Vesikel von nicht-pathogenen Amöben verwendbar für den Transfer eines Moleküles von Interesse in eine eukaryotische Zelle
US7384589B2 (en) 2003-08-01 2008-06-10 Lawrence Livermore National Security, Llc Nanoscale molecularly imprinted polymers and method thereof
CA2453198A1 (en) 2004-01-07 2005-07-07 Wei-Ping Min Quantification and generation of immune suppressive exosomes
US10072297B2 (en) 2004-02-20 2018-09-11 The Regents Of The University Of California Salivary mRNA profiling, biomarkers and related methods
SI1733056T1 (sl) 2004-03-31 2013-10-30 The General Hospital Corporation Postopek določanja odzivnosti rakaste tvorbe na zdravljenje, kjer je tarča receptor epidermalnega rastnega faktorja
US20080268429A1 (en) * 2004-06-02 2008-10-30 Sourcepharm, Inc. Rna - Containing Microvesicles and Methods Therefor
US8021847B2 (en) 2004-06-02 2011-09-20 Proxy Life Science Holdings, Inc. Microvesicle-based compositions and methods
US7867707B2 (en) 2004-06-11 2011-01-11 Evotec Ag Method for detecting analytes in a sample
US20060116321A1 (en) 2004-07-01 2006-06-01 Robbins Paul D Immunosuppressive exosomes
US7347331B2 (en) 2004-08-13 2008-03-25 Regents Of The University Of Minnesota Fines removal apparatus and methods/systems regarding same
AP2007003959A0 (en) 2004-10-07 2007-04-30 Ananda Chakrabarty Cupredoxin derived transport agents and methods ofuse thereof
EP1815021A2 (de) 2004-11-03 2007-08-08 Almac Diagnostics Limited Transkriptom-mikroarray-technologie und verfahren zu ihrer verwendung
US20060223072A1 (en) 2005-03-31 2006-10-05 Boyes Barry E Methods of using a DNase I-like enzyme
EP1869221A2 (de) 2005-04-15 2007-12-26 Cedars-Sinai Medical Center Durchflusszytometrische heteroduplex-analyse zur detektion genetischer veränderungen
EP1883710A4 (de) 2005-05-03 2009-07-08 Althea Technologies Inc Zusammensetzungen und verfahren zur analyse abgebauter nukleinsäuren
WO2007015174A2 (en) 2005-07-08 2007-02-08 Exothera L.L.C. Exosome-specific ligands, their preparartion and uses
EP1904642A4 (de) 2005-07-19 2008-11-26 Univ Illinois Transportmittel zur überquerung der blut-hirn-schranke und zum eindringen in gehirnkrebszellen sowie verwendungsverfahren dafür
WO2007100699A2 (en) 2006-02-24 2007-09-07 Novartis Ag Microparticles containing biodegradable polymer and cationic polysaccharide for use in immunogenic compositions
EP3517151B1 (de) 2006-03-09 2021-04-21 Aethlon Medical, Inc. Extrakorporale entfernung von mikrovesikulären teilchen
WO2007127848A1 (en) 2006-04-26 2007-11-08 University Of Louisville Research Foundation, Inc Isolation of membrane vesicles from biological fluids and methods of using same
US9085778B2 (en) 2006-05-03 2015-07-21 VL27, Inc. Exosome transfer of nucleic acids to cells
CN101085349B (zh) 2006-06-09 2011-05-25 项雯华 囊泡导向的免疫细胞及其在制备抗肿瘤药物上的应用
WO2008084331A2 (en) 2006-06-21 2008-07-17 Hopitaux Universitaires De Geneve Biomarkers for renal disorders
JP2008035779A (ja) 2006-08-07 2008-02-21 Mitsubishi Rayon Co Ltd 核酸の分解度を測定する方法と核酸アレイ
EP1961825A1 (de) 2007-02-26 2008-08-27 INSERM (Institut National de la Santé et de la Recherche Medicale) Verfahren zur Vorhersage des Auftretens von Metastasen bei Brustkrebspatienten
US20080287669A1 (en) 2007-05-16 2008-11-20 Braman Jeffrey C Methods and compositions for identifying compounds useful in nucleic acid purification
EP2167138A2 (de) 2007-06-08 2010-03-31 Asuragen, INC. Mir-34-regulierte gene und pfade als targets für therapeutische intervention
ES2420973T3 (es) 2007-07-25 2013-08-28 University Of Louisville Research Foundation, Inc. Micro-ARN asociado a exosoma como marcador de diagnóstico
CA2733672C (en) 2007-08-16 2018-09-11 The Royal Institution For The Advancement Of Learning/Mcgill University Tumor cell-derived microvesicles
US20100255514A1 (en) 2007-08-16 2010-10-07 The Royal Institution For The Advancement Of Learning/Mcgill University Tumor cell-derived microvesicles
AU2008295394B2 (en) 2007-09-05 2014-04-24 Laurentian University Method of using tumour RNA integrity to measure response to chemotherapy in cancer patients
WO2009036236A1 (en) 2007-09-14 2009-03-19 The Ohio State University Research Foundation Mirna expression in human peripheral blood microvesicles and uses thereof
US8617806B2 (en) 2008-01-25 2013-12-31 Hansabiomed Ou Method to measure and characterize microvesicles in the human body fluids
DK2245199T3 (da) 2008-02-01 2014-02-03 Gen Hospital Corp Anvendelse af mikrovesikler i diagnosticering, prognosticering og behandling af medicinske sygdomme og tilstande
US20120142001A1 (en) 2008-02-01 2012-06-07 Exosome Diagnostics, Inc. Method for isolation of nucleic acid containing particles and extraction of nucleic acids therefrom
US20100008978A1 (en) 2008-05-09 2010-01-14 The Regents Of The University Of California Nanoparticles effective for internalization into cells
CA2728736A1 (en) 2008-06-20 2009-12-23 Proxy Life Science Holdings, Inc. Microvesicle-based compositions and methods
EP2326735B1 (de) 2008-09-03 2016-11-16 The Johns Hopkins University Genetische veränderungen bei isocitrat-dehydrogenase und anderen genen bei malignem gliom
WO2010065968A1 (en) 2008-12-05 2010-06-10 Myriad Genetics, Inc. Cancer detection markers
US20120058480A1 (en) 2009-02-24 2012-03-08 Lewis Dorothy E Antigenic approach to the detection and isolation of microparticles associated with fetal dna
CA3132169A1 (en) * 2009-06-05 2010-12-09 Myriad Genetics, Inc. Methods of detecting cancer comprising screening for mutations in the apc, egfr, kras, pten and tp53 genes
EP2454588B1 (de) 2009-07-16 2015-07-15 The General Hospital Corporation Nukleinsäureanalyse
US20130029339A1 (en) 2009-09-09 2013-01-31 The General Hospital Corporation Use of microvesicles in analyzing kras mutations
EP3461912B1 (de) 2009-09-09 2022-07-13 The General Hospital Corporation Verwendung von mikrovesikeln bei der analyse von nukleinsäureprofilen
CA2787027A1 (en) 2010-01-13 2011-07-21 Caris Life Sciences Luxembourg Holdings, S.A.R.L. Detection of gastrointestinal disorders
KR20130043104A (ko) 2010-04-06 2013-04-29 카리스 라이프 사이언스 룩셈부르크 홀딩스 질병용 순환 생물학적 지표들
US20130040833A1 (en) 2010-05-12 2013-02-14 The General Hospital Corporation Use of microvesicles in analyzing nucleic acid profiles
WO2012031008A2 (en) 2010-08-31 2012-03-08 The General Hospital Corporation Cancer-related biological materials in microvesicles

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110081651A1 (en) * 2005-06-28 2011-04-07 Genentech, Inc. Egfr and kras mutations
US20100184046A1 (en) * 2008-11-12 2010-07-22 Caris Mpi, Inc. Methods and systems of using exosomes for determining phenotypes

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160237422A1 (en) * 2011-05-11 2016-08-18 Exosome Diagnostics, Inc. Nucleic acid extraction from heterogeneous biological materials
US11407991B2 (en) * 2011-05-11 2022-08-09 Exosome Diagnostics, Inc. Nucleic acid extraction from heterogeneous biological materials
US11180812B2 (en) * 2012-08-17 2021-11-23 Cornell University Use of DNA in circulating exosomes as a diagnostic marker for metastatic disease
US20150218651A1 (en) * 2012-08-17 2015-08-06 Cornell University Use of dna in circulating exosomes as a diagnostic marker for metastatic disease
US9958448B2 (en) 2012-10-23 2018-05-01 Caris Life Sciences Switzerland Holdings Gmbh Aptamers and uses thereof
US10942184B2 (en) 2012-10-23 2021-03-09 Caris Science, Inc. Aptamers and uses thereof
US9939443B2 (en) 2012-12-19 2018-04-10 Caris Life Sciences Switzerland Holdings Gmbh Compositions and methods for aptamer screening
US10865451B2 (en) 2013-06-11 2020-12-15 Dana-Farber Cancer Institute, Inc. Non-invasive blood based monitoring of genomic alterations in cancer
WO2014201092A1 (en) * 2013-06-11 2014-12-18 Dana-Farber Cancer Institute, Inc. Non-invasive blood based monitoring of genomic alterations in cancer
US10844436B2 (en) 2014-04-01 2020-11-24 Cornell University Use of double-stranded DNA in exosomes: a novel biomarker in cancer detection
US11397182B2 (en) 2014-10-07 2022-07-26 Cornell University Methods for prognosing and preventing metastatic liver disease
WO2016077709A1 (en) * 2014-11-14 2016-05-19 Liquid Genomics, Inc. Use of circulating cell-free rna for diagnosis and/or monitoring cancer
US11773447B2 (en) 2014-11-14 2023-10-03 Nanthealth Labs, Inc. Use of circulating cell-free RNA for diagnosis and/or monitoring cancer
US11899024B2 (en) 2017-07-12 2024-02-13 Exosome Diagnostics, Inc. Treatment and diagnosis of parkinson's disease using isolated and enriched populations of biofluid-derived extracellular vesicles

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