[go: up one dir, main page]

WO2014160032A1 - Arn associé à un exosome en tant que marqueur de diagnostic - Google Patents

Arn associé à un exosome en tant que marqueur de diagnostic Download PDF

Info

Publication number
WO2014160032A1
WO2014160032A1 PCT/US2014/025675 US2014025675W WO2014160032A1 WO 2014160032 A1 WO2014160032 A1 WO 2014160032A1 US 2014025675 W US2014025675 W US 2014025675W WO 2014160032 A1 WO2014160032 A1 WO 2014160032A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
rnas
microvesicles
incrnas
kit
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/025675
Other languages
English (en)
Inventor
Douglas Taylor
Cicek Gercel-Taylor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Louisville Research Foundation ULRF
Original Assignee
University of Louisville Research Foundation ULRF
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Louisville Research Foundation ULRF filed Critical University of Louisville Research Foundation ULRF
Publication of WO2014160032A1 publication Critical patent/WO2014160032A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the presently-disclosed subject matter relates to characterization of diseases and evaluation of treatment and/or progression of diseases based on determining amounts of one or more exosome-derived RNAs in a biological sample.
  • the presently-disclosed subject matter relates to determining amounts of one or more exosome-derived long non- coding RNAs (IncRNAs) and/or microRNAs (miRs or miRNAs).
  • ovarian cancer remains the fourth leading cause of cancer-related deaths in women, resulting in more than 25,500 new cases and 15,310 deaths annually in the U.S. 1 2
  • Most women with ovarian cancer are diagnosed at an advanced stage, with 75% of cases diagnosed with extra-ovarian disease. This late diagnosis may reflect the inaccessibility of the ovaries and the lack of early symptoms.
  • 3 The anatomical location of the ovaries results in minimal interference with vital structures or local irritation, making the diagnosis of ovarian cancer difficult, until regional and distant metastases have occurred.
  • ovarian cancer accounts for only a third of gynecologic cancers, it results in 55% of the deaths from gynecologic malignancies and 6% of all cancer deaths in women. 4 ' 5 Long-term survival has not changed significantly in the last two decades, largely due to inadequacy of diagnostic approaches, which only detects well-established overt cancers.
  • Stage I ovarian cancer can be cured in 90% of cases, while five-year survival for patients with advanced disease (Stage III and IV) is less than 21%.
  • Stage III and IV advanced disease
  • 73% of endometrial cancers, 55% of breast cancers and 50% of cervical cancers are diagnosed as Stage I, while only 23% of ovarian cancers are diagnosed at an early stage. 6
  • prospects for significant improvement in ovarian cancer survival reside in early diagnosis of disease.
  • CA125 The only biomarker currently approved for ovarian cancer detection is CA125 and its quantitation by ELISA has been the "gold standard" for detection of ovarian cancer, since its introduction in 1983. 7 Assessment of CA125 is typically used in disease management, both for disease detection as well as monitoring for disease recurrence;
  • CA125 is neither sensitive nor specific for de novo ovarian cancer detection, since it is elevated in >50% of women with stage I disease, although it is elevated in more than 80% of patients with advanced stage ovarian cancer.
  • CA125 has poor specificity, which is shown by its elevation in association with benign and malignant breast and colon disease, peritoneal irritants, and benign gynecologic diseases, among others. Significant effort has been expended in the recent years for identifying potential markers that might substitute or complement CA125 in disease management or ultimately in the design of screening strategies.
  • SELDI-TOF-MS profiling has been successfully used to differentiate ovarian, breast, prostate, and liver cancer from controls. 10 SELDI-TOF- MS profiling of serum was significantly better than the current standard serum biomarker CA125 at distinguishing patients with ovarian cancer from those with benign ovarian disease and from healthy controls. 11 Studies have shown that the selection of a combination of multiple proteins resolved by SELDI-TOF-MS may become a potential diagnostic approach. An effective screening test for ovarian cancer needs to achieve a high sensitivity and specificity and currently, different proteomic technologies as well as the computational analytic tools used to discern peaks generate different findings. These initial studies on SELDI-TOF-MS profiling insights are promising, and the concept is reproducible in a series of different backgrounds; however, translating this approach into a routine diagnostic test remains difficult.
  • Non-small-cell lung carcinoma comprises any epithelial cancer of the lung other than small-cell lung carcinoma.
  • Lung cancers are mainly observed in tobacco smokers.
  • NSCLCs are primarily treated by surgical resection.
  • neoadjuvant and adjuvant chemotherapy is becoming more common, e.g., cisplatin.
  • Radiation therapy is also used.
  • Chemotherapy is used more often for metastatic disease, e.g., EGFR tyrosine kinase inhibitors such as gefitinib.
  • the most common types of NSCLC are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. Adenocarcinomas are the most common type of lung cancer in non-smokers.
  • Figure 1 NTA profile of unfractionated circulating vesicles in representative analyses of normal female controls, patients with benign ovarian disease and patients with ovarian cancer. Sera were diluted in PBS and analyzed using a Nanosight LM10. Inset values represent the total number of vesicles counted/ml. The bar graph presents the mean and standard deviation of the vesicles concentrations for patients with benign and malignant ovarian disease.
  • FIG. 2 Chromatographic isolation of circulating extracellular vesicles.
  • Panel A A representative chromatogram of a serum sample obtained from ovarian cancer patient TB08-36 fractionated using a 2% agarose-based size exclusion gel. The peak containing exosomes appears in the void volume.
  • Panel B The three fractions corresponding to the void volume of the column were diluted in PBS and analyzed by NTA using a Nanosight LM10.
  • Figure 3 Electrophoretic analyses of the chromatographic fractions from ovarian cancer patient TB08-36 and patient with benign adenoma.
  • Panel A The separation of proteins from the chromatographic fraction on SDS-PAGE followed by total protein staining with Imperial Purple. Imperial purple stained 10% SDS-PAGE analysis of a comparable amount of proteins (25 ⁇ g).
  • Panel B Western immunoblot evaluation of published markers of exosomes (CD63), microvesicles (CD154) and tumor origin
  • EGFRvIII in vesicles isolated from patient TB08-36.
  • Panel C Western immunoblot evaluation of published markers of exosomes (CD63) and tumor origin (EGFRvIII) in vesicles isolated from patient with benign disease. Chromatographically derived vesicles (25 ⁇ g protein) were separated on a 10% SDS-PAGE, transferred to nitrocellulose membranes, incubated with anti-CD63, anti-CD 154, or anti -EGFRvIII antibodies.
  • Figure 4 Comparison of vesicle analyses by electron microscopy (A), NTA (B), DLS (C) and submicron particle analysis (D).
  • EM images reveal the presence of nano-sized vesicles with a circular shape.
  • Scale bar 100 nm (original magnification *45K).
  • Figure 5 Combination of NTA and fluorescent antibodies to characterize the phenotypes of chromatographically isolated vesicles from two ovarian cancer patients.
  • Vesicle suspensions were incubated with either Qdot-labeled anti-CD63 or Odot-labeled antiEpCAM. These vesicles were then examined in light scattering mode to define total vesicle size distribution (Panel A) or in fluorescence mode to define CD63- positive vesicles (Panel B) or EpCAM- positive vesicles (Panel C).
  • Figure 6 Disruption of light scattering defined particles by non-ionic detergents.
  • the size distributions of chromatographically isolated vesicles were analyzed by either NTA (A) or submicron particle analysis (C).
  • the same vesicles suspension was treated with either 0.5% Triton X-100 (B) or 1% Tween 20 (D) for 5 minutes at room temperature and re-analyzed.
  • Treatment with non-ionic detergents disrupted the vesicles, either reducing the number and size under NTA or producing a size shift by SPA.
  • FIG. 7 Presence of specific miRNA in tumor derived exosomes (versus normal controls). miRNAs shown to the left of the red line have been demonstrated to be down- regulated in ovarian tumor cells while miRNAs to the right of the vertical line have been shown to be up-regulated in ovarian cancer.
  • Figure 8 Heat maps of microRNA arrays examining the expression of miRNA in exosomes isolated from ovarian cancer patients at various stages versus controls.
  • Figure 9 Scatter plot of miRNA expression associated with exosomes from Stage I (Group 1), Stage II (Group 2) or Stage III (Group 3) ovarian cancer
  • Figure 10 Association of specific miRNAs with stages of ovarian cancer.
  • Control lane represents miRNA isolated from normal controls.
  • Group 1 represents exosomal miRNAs isolated from Stage I,
  • Group 2 corresponds to Stage II and Group 3 to Stage III.
  • Figure 11A Association of long-noncoding RNA with exosomes from ovarian cancer patients (versus controls).
  • Figure 11B Association of long-noncoding RNA with exosomes from lung cancer patients (versus controls).
  • Figure 12 Scatter plot showing the comparison of microRNAs expressed in pregnant patient exhibiting spontaneous pregnancy loss (Group 1) versus these pregnancies continuing to term (control)
  • Figure 13 Heat map of microRNA expression observed in exosomes from patients experiencing recurrent pregnancy loss (RPL) versus normal term delivering pregnancies.
  • Figure 14 Pathway of genes regulated by specific microRNAs associated with exosomes isolated from women experiencing recurrent pregnancy loss.
  • Figure 15 Pathway induced by genes regulated by microRNAs associated with exosomes derived from pregnant women experiencing recurrent pregnancy loss.
  • MicroRNAs small (e.g., 17-25 nucleotides in length) non-coding RNAs, suppress the translation of target mRNAs by binding to their 3 ' untranslated region (Esquela- Kerscher & Slack, 2006; Bartel, 2004). Post-transcriptional silencing of target genes by miRNA can occur either by cleavage of homologous mRNA or by specific inhibition of protein synthesis.
  • miRNA-200a and miR-200c Two of these up-modulated miRNAs, miR-200a and miR-200c, were enhanced in all the three histologic types examined (serous, endometrioid, and clear cell), whereas miR-200b and miR-141 up-modulation was shared by endometrioid and serous histologic types.
  • miRNA signatures obtained comparing different histologic types of ovarian cancers (serous, endometrioid, clear cell, and mixed) with the normal tissue were overlapping in most cases.
  • Their analysis of ovarian tumors also demonstrated the absence of differentially expressed miRNAs in relation to tumor stage or grade, which could have resulted from their set of samples being primarily derived from advanced stage tumors.
  • miR-200a and miR-141 belong to the same family, miR-200b is localized on chromosome lp36.33 in the same region as miR-200a and miR-200c is localized on chromosome 12p 13.31 in the same region of miR- 141 (Iorio et al. (2007)). This association would agree with the findings of Zhang et al.
  • miRNA expression patterns appear to be more characteristic of the developmental origins of tumors than mRNA expression patterns and may be associated with diagnosis, staging, progression, prognosis, and response to treatment.
  • analyses of miRNA signatures prior to the presently-disclosed subject matter, the analyses of miRNA signatures have been limited to tissue biopsies.
  • tumors One general characteristic of tumors is their ability to release or shed intact, vesicular portions of the plasma membrane (termed membrane fragments, membrane vesicles, microvesicles or exosomes), which was initially described by the present inventors. 13 "Exosomes" are microvesicles released from a variety of different cells, including cancer cells (i.e., "cancer-derived exosomes”). These small vesicles (50-100nm in diameter), which were present inside large multivesicular endosomes, contained transferrin receptors, a marker that is used to follow endocytosis and the recycling of cell-surface proteins, that had been internalized from the plasma membrane.
  • microvesicle is inclusive of microparticles known in the art as exosomes and microvesicles.
  • exosomes and microvesicles The precise mechanisms of exosome release/shedding remain unclear; however, this release is an energy-requiring phenomenon, modulated by extracellular signals. They appear to form by invagination and budding from the limiting membrane of late endosomes, resulting in vesicles that contain cytosol and that expose the extracellular domain of transferrin receptors at their surface.
  • Using electron microscopy studies have shown fusion profiles of multivesicular endosomes with the plasma membrane, leading to the secretion of the internal vesicles into the extracellular environment. The rate of exosome release is significantly increased in most neoplastic cells and occurs continuously.
  • exosomes and their accumulation appear to be important in the malignant transformation process.
  • the release of exosomes was also demonstrated to be associated with cells of embryonic origin (such as the placenta) and activated lymphoid cells. 17"20
  • extracellular shedding of exosomes occurs in other types of cells, under specific physiological conditions, the accumulation of exosomes from non-neoplastic cells is rarely observed, in vivo 1 ' 21
  • exosomes released by tumor cells accumulate in biologic fluids, including in sera, ascites, and pleural fluids. Exosome release and its accumulation appear to be important features of the malignant transformation.
  • Shed tumor derived exosomes do not mirror the general composition of the plasma membrane of the originating tumor cell, but represent 'micromaps,' with enhanced expression of tumor antigens. 17 ' 22
  • exosome release by tumor cells is a re-expression of the fetal cell exosomes and that both constituted pathways to circumvent
  • miRNAs are small endogenous noncoding RNA gene products about 22 nucleotides (nt) long that regulate gene expression in a sequence-specific manner and are found in diverse organisms. With >300 already identified, the human genome may contain up to 1,000 miRNAs. miRNA play key roles in regulating the translation and degradation of messenger RNAs through base pairing to partially complementary sites, predominately in the untranslated region of the message. 31"33 miRNAs are expressed as long precursor RNAs. Drosha, an RNase III endonuclease, is responsible for processing primary miRNAs in the nucleus and releasing ⁇ 70nt precursor miRNAs.
  • Drosha an RNase III endonuclease
  • Drosha associates with the dsRNA -binding protein DGCR8 in human to form the microprocessor complex.
  • Precursor miRNAs are transported to the cytoplasm by exportin-5 and cleaved by the RNase III endonuclease Dicer, releasing 17-24nt mature dsmiRNA.
  • 36 ' 37 One strand of the miRNA duplex is subsequently incorporated into the effector complex RNA-induced silencing complex (RISC) that mediates target gene expression.
  • RISC RNA-induced silencing complex
  • Argonaute2 a key component of RISC, may function as an endonuclease that cleaves target mRNAs.
  • miRNAs While the biological functions of most miRNAs are not yet fully understood, it has been suggested that the miRNAs are involved in various biological processes, including cell proliferation, cell death, stress resistance, and fat metabolism, through the regulation of gene expression. 38 As potential clinical diagnostic tools miRNAs have been shown to be important and accurate determinants for many if not all cancers. 39 Increasing evidence shows that expression of miRNA genes is deregulated in human cancer. The expression of miRNAs is highly specific for tissues and developmental stages and has allowed recently for molecular classification of tumors. To date, all tumors analyzed by miRNA profiling have shown significantly different miRNA profiles compared with normal cells from the same tissue.
  • miRNA-expression profiles classify human cancers according to the developmental lineage and differentiation state of the tumors. Specific over- or underexpression has been shown to correlate with particular tumor types. miRNA overexpression could result in down-regulation of tumor suppressor genes, whereas their underexpression could lead to oncogene up-regulation.
  • cancer cells showed distinct miRNA profiles compared with normal cells with 36 of the 228 miRNA genes overexpressed and 21 downregulated in cancer cells versus normal cells. 40 Hierarchical clustering analyses showed that this miRNA signature enabled the tumor samples to be grouped on the basis of their tissue of origin.
  • Genome-wide profiling studies have been performed on various cancer types, including CLL, 41 breast cancer, 42 glioblastoma, 43 thyroid papillary carcinoma, 44 hepatocellular carcinoma, 45 ovarian cancer, 46 colon cancer, 47 and endocrine pancreatic tumours.
  • 48 In a study of 104 matched pairs of primary cancerous and non-cancerous ovarian tissue, 43 differentially expressed miRNAs were observed; 28 were downregulated and 15 were overexpressed in tumors.
  • Statistical analyses of microarray data obtained by two different methods significance analysis of microarrays (SAM) and prediction analysis of microarrays (PAM) from six solid tumors (ovarian, breast, colon, gastric and prostate carcinomas and endocrine pancreatic tumors), demonstrated a common signature composed of 21 miRNAs differentially expressed in at least three tumor types. 50 At the top of the list were miR-21, which was overexpressed in six types of cancer cells, and miR-17-5p and miR-191, which were overexpressed in five. As the embryological origin of the analyzed tumors was different, the significance of such findings could be that these common miRNAs participate in fundamental signaling pathways altered in many types of tumor.
  • the present inventors have observed that while some miRNAs that are up-regulated with the tumor (i.e., identified using tumor biopsies or cultured tumor cells) are also up-regulated in their exosomes, some tumor-up-regulated miRNAs are not up- regulated within exosomes.
  • certain miRNAs that exhibit down-regulation within the tumor are up-regulated in exosomes.
  • miRNA signatures derived from ovarian tumor cell exosomes exhibit some miRNAs that are undetectable within the tumor.
  • LncRNAs Long noncoding RNAs
  • Dysregulation of IncRNA expression has been shown to be associated with a wide range of defects in development and pathologies.
  • IncRNAs have been found to exhibit a wide range of functions ranging from signaling, serving as molecular decoys, guiding ribonulceoprotein complexes to specific chromatin sites and also participating as scaffolds in the formation of complexes.
  • IncRNAs The transcription of certain IncRNAs is very tissue and temporal specific. Their expression can be in response to certain stimuli, such as cellular stress and temperature. Thus, IncRNAs can serve as molecular signals and can act as markers of functionally significant biological events. Decoys: The molecular decoy type of activity takes place when specific IncRNAs are transcribed and then bind to and titrate away protein factors. Decoy IncRNAs can "sponge" protein factors such as transcription factors and chromatin modifiers. This leads to broad changes in the cell's transcriptome. Guides:
  • IncRNAs can be molecular guides by localizing particular ribonucleoprotein complexes to specific chromatin targets. This activity can cause changes in gene expression either in cis (on neighboring genes) or in trans (distantly located genes) that cannot be easily predicted by just the IncRNA sequence itself. Scaffolds: Assembly of complex protein complexes can be supported by IncRNAs, linking factors to together to form new functions. LncR As function as molecular scaffolds regulating histone modifications and influence the epigenetic programs of the transcriptome. Some IncRNAs possesses different domains that bind distinct protein factors that altogether, may impact transcriptional activation or repression.
  • IncRNAs have been identified within cells, the present inventors have demonstrated their presence within circulating exosomes. The presence of exosomal lnc RNA is demonstrative of the presence of ovarian disease, with specific patterns distinguishing benign and malignant pathologies.
  • miRNA control levels are presently disclosed subject matter.
  • the presently disclosed subject matter further discloses that long non-coding RNA (IncRNA) isolated from cancer-derived microvesicles exhibits expression levels in subjects suffering from cancer that differ (e.g., increased or decreased) from IncRNA expression levels measured in subjects free of cancer (referred to herein as "IncRNA control levels").
  • IncRNA control levels long non-coding RNA
  • the presently disclosed subject matter provides for the isolation of cancer- derived microvesicles from readily-accessible biological fluids from a test subject. As such, the presently disclosed subject matter provides methods for diagnosis and prognosis of cancer based on the collection and measurement of cancer-derived microvesicle RNA levels from readily-accessible biological samples, and without necessitating direct sampling of cancer cells.
  • RNAs of a disease e.g., a RNA signature or RNA expression profile
  • the method involves isolating disease-derived microvesicles from a sample, isolating IncRNA from said disease-derived microvesicles, and determining a presence of one or more IncRNAs in said disease-derived microvesicles.
  • determining the presence of one or more RNAs includes determining an expression profile of the one or more RNAs.
  • the one or more RNAs in the sample or the expression profile of the one or more RNAs in the sample can be compared to a reference.
  • the sample can be a biological sample obtained from a subject.
  • the sample can be obtained from a cell culture.
  • a method for characterizing a disease in a subject is provided. Characterizing can include providing a diagnosis, prognosis, and/or theragnosis of the disease. In some embodiments, the method can include isolating disease-derived microvesicles from a biological sample of the subject, determining an amount of one or more RNAs in the isolated disease-derived microvesicles, and comparing the amount of the one or more RNAs to a reference, wherein the disease is characterized based on a measurable difference in the amount of the one or more RNAs from the disease-derived microvesicles as compared to a control. For example, in some embodiments the subject can be diagnosed as having the disease or risk thereof if there is a measurable difference in the amount of the one or more RNAs from the disease-derived microvesicles in the sample as compared to a reference.
  • a method for evaluation treatment efficacy and/or progression of a disease in a subject can involve isolating disease-derived microvesicles from a biological sample of the subject, determining an amount of one or more RNAs in the isolated disease-derived microvesicles, and determining any measurable change in the amounts of the one or more RNAs to thereby evaluate treatment efficacy and/or progression of the cancer in the subject.
  • the biological sample can include a first biological sample collected prior to initiation of treatment for the disease and/or onset of the disease and a second biological sample collected after initiation of the treatment or onset.
  • the method can also include selecting a treatment or modifying a treatment for the disease based on the amount of the one or more RNAs determined.
  • the disease of interest can be, for example, a cancer.
  • the disease-derived microvesicles are cancer-derived microvesicles.
  • the cancer is a ovarian cancer.
  • the cancer is a lung cancer.
  • the disease of interest can be a pregnancy outcome.
  • the disease-derived microvesicles are placenta-derived microvesicles.
  • cancer refers to all types of cancer or neoplasm or malignant tumors found in animals, including leukemias, carcinomas, adenomas and sarcomas.
  • Examples of cancers are cancer of the brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, pancreas, prostate, sarcoma, stomach, and uterus.
  • leukemia includes progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere
  • nasopharyngeal carcinoma oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcom
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • squamous cells refers to the epithelium (tissue layer) that is the surface cells of much of the body.
  • skin and mucous membranes are squamous cells.
  • Squamous cell neoplasms include without limitation papillary carcinoma, verrucous squamous cell carcinoma, papillary squamous cell carcinoma, squamous cell carcinoma, large cell keratinizing squamous cell carcinoma, small cell keratinizing squamous cell carcinoma, spindle cell squamous cell carcinoma, adenoid/pseudoglandular squamous cell carcinoma, intraepidermal squamous cell carcinoma, lymphoepithelial carcinoma, basaloid squamous cell carcinoma, clear cell squamous cell carcinoma, keratoacanthoma, signet ring cell squamous cell carcinoma, and spindle cell squamous cell carcinoma.
  • Squamous cell carcinoma is one of the most common cancers in humans, and usually arises from mutated ectodermal or endodermal cells lining body cavities. It can develop in a variety of organs and tissues, including the skin, lips, mouth, esophagus, urinary bladder, prostate, lung, vagina, cervix, and others. Squamous cell carcinoma is most likely to appear in males over 40 years of age with a history of heavy alcohol use coupled with smoking. Head and neck squamous cell carcinoma (HNSCC) is the most common form of larynx cancer, accounting for over 90% of throat cancer. Squamous cell lung carcinoma is a type of non-small-cell lung carcinoma ( SCLC) and is closely correlated with a history of tobacco smoking.
  • SCLC non-small-cell lung carcinoma
  • a method for characterizing an ovarian cancer or a lung cancer in a subject includes isolating microvesicles from a biological sample of the subject; determining a presence or an amount of one or more RNAs from the isolated microvesicles; and comparing the presence or the amount of the one or more RNAs to a reference, wherein the ovarian cancer or lung cancer is characterized based on a measurable difference in the presence or the amount of the one or more RNAs from the isolated microvesicles as compared to the reference.
  • the characterizing comprises providing a diagnosis, prognosis and/or theragnosis of the cancer.
  • a method for evaluating treatment efficacy and/or progression of a ovarian cancer or lung cancer in a subject includes isolating microvesicles from a biological sample of the subject; determining a presence or an amount of one or more RNAs in the isolated microvesicles; and comparing the presence or the amount of the one or more RNAs to a reference, wherein the treatment efficacy and/or progression of the ovarian cancer or lung cancer is evaluated based on a measurable difference in the presence or the amount of the one or more RNAs as compared to the reference.
  • a method for assessing the presence of one or more RNAs of a lung cancer RNA signature or an ovarian cancer RNA signature includes isolating cancer-derived, extracellular microvesicles from a biological sample; and determining a presence of one or more RNAs in said microvesicles.
  • the microvesicles are shed from lung cancer or ovarian cancer cells.
  • a method for characterizing a pregnancy outcome in a subject is provided.
  • circulating exosomes derived from the placenta can be isolated from a biological sample, such as for example blood, or components thereof.
  • the placenta while derived from the fetus, is the only fetal tissue actually in contact with the maternal system. As such, exosomes produced by placental cells can circulate within the bloodstream of the mother.
  • anti-EpCAM antibodies as used for tumor exosome isolation
  • PLAP anti-placental type alkaline phosphatase antibodies
  • the method comprises providing a biological sample from a subject and isolating exosomes comprising RNAs from the biological sample. An amount of one or more of the RNAs is then determined and compared to a reference. The subject can then be diagnosed with being at risk for an adverse pregnancy outcome if there is a measurable difference in the amount of the one or more miRNAs from the exosomes as compared to the reference.
  • the adverse pregnancy outcome is a disorder selected from the group consisting of preeclampsia, preterm birth (e.g., delivery before 32 weeks gestation), premature rupture of membranes, intrauterine growth restriction, and recurrent pregnancy loss.
  • methods of the presently-disclosed subject matter include determining an expression profile or a signature of two or more RNAs.
  • the methods can include comparing the expression profile with a profile from a selected reference sample to determine the presence or the amount of two or more RNAs in said microvesicles.
  • a biomarker expression profile or biomarker signature for a sample can include information about the identities of biomarkers contained in the sample, quantitative levels of biomarkers contained in the sample, and/or changes in quantitative levels of biomarkers relative to another sample or control.
  • a biomarker signature or profile for a sample can include information about the identities, quantitative levels, and/or changes in quantitative levels of biomarkers from an cancer-derived extracellular microvesicles from a biological sample of particular subject.
  • a biomarker signature or profile relates to information about two or more biomarkers in a sample (e.g., biomarker signature or profile consisting of 2 biomarkers).
  • a biomarker signature or profile consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 biomarkers.
  • the one or more RNAs measured can be selected from long non-coding RNAs (IncRNAs) and/or miRNAs. These RNAs can be measured to detect a disease, such as a pregnancy outcome or a cancer such as those described herein, e.g., ovarian cancer and/or a lung cancer.
  • the cancer comprises squamous cell carcinoma.
  • methods of the presently-disclosed subject matter can be performed in vitro.
  • methods of the presently-disclosed subject matter can be performed ex vivo.
  • RNAs that can be measured include miRNAs, including those miRNAs as set forth in the miRBase Registry, and IncRNAs, including those IncRNAs as set forth in the IncRNAdb.
  • IncRNAs examples include 21 A, 7SL, Alpha 280, BACElAs, E2F4 antisense, H19 antisense, SFMBT2, Tsix, RoR, Y-RNA-1, HAR1A, HAR- 1B, Air, Malat7, and Y RNA-1. Further examples of IncRNAs that can be measured include IncRNAs comprising the sequence of SEQ ID NOs: 1-9.
  • IncRNAs comprising a sequence at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to any one of SEQ ID NO: 1-9.
  • percent identity or “percent homology” when used herein to describe to a sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873- 5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990).
  • BLAST basic local alignment search tool
  • Gapped BLAST is utilized as described in Altschul, et al. (Nucleic Acids Res. 25: 3389-3402, 1997).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST are used. See http://www.ncbi.nlm.nik.gov.
  • miRNAs that can be measured include let7a, miR222, let7b, miRl 00, miR155, miR9, miR125b, and miR203. Further examples of miRNAs that can be measured include let 7a, miR-122, miR-142, miR-96, miR222, miR- 148b. Further examples of miRNAs that can be measured include miR142-3p, 520, 130a, 222,452, 488, 215, 206, 498, 302c, 133b, and 142- 5p.
  • miRNAs that can be measured include of miR 10b, 1, 101, 370, 21, 223, 20a, 15b, 24, 99a, 146a, 302a, 195, 23b, 16, 26a, 182, 146b-5p, 185, 7, 424,194,96, and 100, and let 7b, let7i, let7a, and let7c.
  • miRNAs that can be measured include miRl 42- 3p, 520, 130a, 222,452, 488, 215, 206, 498, 302c, 133b, 142-5p, miR 10b, 1, 101, 370, 21, 223, 20a, 15b, 24, 99a, 146a, 302a, 195, 23b, 16, 26a, 182, 146b-5p, 185, 7, 424,194,96, 100, and let 7b, let7i, let7a, and let7c.
  • miRNAs that can be measured include those identified in U.S. Patent No. 8,216,784, U.S. Patent Application Serial Nos.12/71 1,499 and 13/489,686, and International Patent Application Serial No. PCT/US12/49615, each of which is incorporated herein by reference.
  • the one or more RNAs include one or more microRNAs selected from the group consisting of: let7a, miR222, let7b, miRl 00, miRl 55, miR9, miRl 25b, and miR203. In some embodiments, the one or more RNAs include one or more microRNAs selected from the group consisting of: let 7a, miR-122, miR-142, miR-96, miR222, miR-148b.
  • the one or more RNAs include one or more IncRNAs selected from the group consisting of: 21A, 7SL, Alpha 280, BACElAs, E2F4 antisense, H19 antisense, SFMBT2, Tsix, RoR, Y-RNA-1, HAR1A, and HAR-1B.
  • the one or more RNAs include one or more IncRNAs comprising the sequence of any one of SEQ ID NOs: 1-9.
  • the one or more RNAs include one or more IncRNAs comprising a sequence having at least75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to any one of SEQ ID NO: 1-9.
  • a change in expression of the one or more RNAs as compared to the reference indicates the presence of ovarian cancer in the subject.
  • the one or more RNAs include one or more IncRNAs selected from the group consisting of: Air, Malat7, and Y RNA-1.
  • the one or more RNAs include IncRNAs comprising the sequence of SEQ ID NOs: 7.
  • the one or more RNAs include one or more IncRNAs comprising a sequence having at least75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to SEQ ID NO: 7.
  • a change in expression of the one or more RNAs as compared to the reference indicates the presence of lung cancer in the subject.
  • the one or more RNAs include one or more microRNAs selected from the group consisting of: miR142-3p, 520, 130a, 222,452, 488, 215, 206, 498, 302c, 133b, and 142-5p.
  • the one or more RNAs include one or more microRNAs selected from the group consisting of: miR 10b, 1, 101, 370, 21, 223, 20a, 15b, 24, 99a, 146a, 302a, 195, 23b, 16, 26a, 182, 146b-5p, 185, 7, 424,194,96, and 100, and let 7b, let7i, let7a, and let7c.
  • the one or more RNAs include one or more microRNAs selected from the group consisting of: miR142-3p, 520, 130a, 222,452, 488, 215, 206, 498, 302c, 133b, 142-5p, miR 10b, 1, 101, 370, 21, 223, 20a, 15b, 24, 99a, 146a, 302a, 195, 23b, 16, 26a, 182, 146b-5p, 185, 7, 424,194,96, 100, and let 7b, let7i, let7a, and let7c.
  • a change in expression of the one or more RNAs as compared to the reference is indicative of an adverse pregnancy outcome in the subject.
  • the terms treatment or treating relate to any treatment of a disease of interest, including but not limited to prophylactic treatment and therapeutic treatment.
  • the terms treatment or treating include, but are not limited to: preventing a disease of interest or the development of a disease of interest; inhibiting the progression of a disease of interest; arresting or preventing the development of a disease of interest; reducing the severity of a disease of interest; ameliorating or relieving symptoms associated with a disease of interest; and causing a regression of the disease of interest or one or more of the symptoms associated with the disease of interest.
  • a method includes comparison to a reference.
  • the reference can include, for example, a level of the one or more RNAs (e.g., miRNAs or lncR As) in one or more samples from one or more individuals without the disease.
  • the reference includes a level of the one or more RNAs in a sample from the subject taken over a time course.
  • the reference includes a sample from the subject collected prior to initiation of treatment for the disease and/or onset of the disease and the biological sample is collected after initiation of the treatment or onset of the disease.
  • the reference can include a standard sample.
  • a standard sample can be a reference that provides amounts of one or more RNAs (e.g., miRNAs or lncRNAs) at levels considered to be control levels.
  • a standard sample can be prepared with to mimic the amounts or levels of one or more RNAs in one or more samples (e.g., an average of amounts or levels from multiple samples) from one or more individuals without the disease of interest.
  • the standard sample can be a reference that provides amounts of one or more RNAs at levels considered to associated with a particular type of cancer and/or a responder or non-responder to treatment.
  • control data when used as a reference, can comprise compilations of data, such as may be contained in a table, chart, graph, e.g., standard curve, or database, which provides amounts or levels of one or more RNAs (e.g., miRNAs or lncRNAs) considered to be control levels.
  • RNAs e.g., miRNAs or lncRNAs
  • Such data can be compiled, for example, by obtaining amounts or levels of one or more RNAs in one or more samples (e.g., an average of amounts or levels from multiple samples) from one or more individuals without the cancer of interest.
  • biomolecule sample refers to a sample that comprises a biomolecule and/or is derived from a subject.
  • biomolecules include, but are not limited to total DNA, RNA, miRNA, mRNA, and polypeptides.
  • the biological sample can be used for the detection of the presence and/or expression level of a miRNA of interest associated with cancer-derived microvesicles. Any cell, group of cells, cell fragment, or cell product can be used with the methods of the presently claimed subject matter, although biological fluids and organs that would be predicted to contain cancer-derived microvesicles exhibiting differential expression of miRNAs as compared to normal controls are best suited.
  • the biological sample is a relatively easily obtained biological sample, such as for example blood or a component thereof.
  • the biological sample comprises milk, blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebral spinal fluid, tears, urine, saliva, sputum, or combinations thereof.
  • the disease- derived microvesicles are isolated using size exclusion chromatography, PEG-precipitation of the microvesicles, filtration, or immunosorbent capture.
  • isolating the microvesicles comprises using an agarose-based gel. Size exclusion chromatography, PEG- precipitation, filtration, and immunosorbent capture techniques are known in the art.
  • a void volume fraction is isolated and comprises the microvesicles of interest.
  • the disease-derived microvesicles can be further isolated after chromatographic separation by centrifugation techniques (of one or more chromatography fractions), as is generally known in the art.
  • centrifugation techniques of one or more chromatography fractions
  • density gradient centrifugation can be used to further isolate the microvesicles.
  • affinity selection refers to the selection of a particular ligand, molecule, substance, or the like based on its affinity for a particular molecule.
  • affinity selection comprises a method for selecting, and thereby isolating, particular microvesicles based on their affinity for particular binding agents.
  • binding agent is used herein to refer to any agent that has known binding affinities.
  • a binding agent can be an antibody or an antibody
  • binding agents can be used in affinity selection to select particular ligands, molecules, substances, or the like based on the extent to which they bind with a particular binding agent.
  • affinity selection comprises separating the cancer- derived microvesicles from non-cancer-derived microvesicles by immunosorbent capture using an anti-cancer antigen antibody as the binding agent.
  • the disease-derived microvesicles are isolated from the sample using a microvesicle surface marker.
  • the surface marker is selected from a group consisting of EpCAM, Fas ligand, PD-1, MICA/B, mdrl, MMPs, CD44, autoreactive antigens, a tetraspanin, and an MHC class I molecule.
  • isolating the microvesicles comprises affinity selection using a binding agent to a microvesicle surface antigen.
  • the microvesicle surface antigen is a known cancer marker.
  • the binding agent is an anti-epithelial cell adhesion molecule (anti-EpCAM) antibody, an anti-CD9 antibody, or an anti-CD63 antibody.
  • a biomarker e.g., an miRNA and/or lncRNA expression level
  • the amount including presence or absence of which is indicative of the presence, severity, or absence of the condition.
  • cancer prognosis is also an area of great concern and interest. It is important to know the aggressiveness of the cancer cells and the likelihood of tumor recurrence in order to plan the most effective therapy.
  • Some cancers are managed by several alternative strategies. In some cases local -regional and systemic radiation therapy is used while in other cases surgical intervention and/or chemotherapy are employed.
  • Current treatment decisions for individual cancer subjects can be based on (1) the number of lymph nodes involved with disease, (2) cancer marker(s) status, (3) the size of the primary tumor, and (4) stage of disease at diagnosis. However, even with these factors, accurate prediction of the course of disease for all cancer subjects is not possible.
  • RNA levels disclosed herein can be useful in order to categorize subjects according to advancement of cancer who will benefit from particular therapies and differentiate from other subjects where alternative or additional therapies can be more appropriate. Treatment related diagnostics are sometimes referred to as "theranosics.” As such, in some embodiments of the presently disclosed subject matter, a method for characterizing a cancer in a subject is provided.
  • the method comprises providing a biological sample from a subject; isolating cancer-derived microvesicles comprising RNAs from the biological sample; determining an amount of one or more of the RNAs; and comparing the amount of the one or more RNAs to one or more RNA control levels.
  • the cancer can be characterized based on a measurable difference in the amount of the one or more RNAs from the cancer-derived microvesicles as compared to the one or more RNA control levels.
  • characterizing the cancer comprises determining a type, a grade, and/or a stage of the cancer.
  • RNA levels can be referred to as “theranosis.”
  • multiple determination of amounts of one or more RNAs over time can be made to facilitate diagnosis (including prognosis), evaluating treatment efficacy, and/or progression of a disease.
  • a temporal change in one or more disease-derived microvesicle RNA levels can be used to predict a clinical outcome, monitor the progression of the disease, and/or efficacy of administered disease therapies.
  • RNA amounts in a biological sample can be used to predict a clinical outcome, monitor the progression of the disease, and/or efficacy of administered disease therapies.
  • the presently disclosed subject matter further provides in some embodiments a method for theranostic testing, such as evaluating treatment efficacy and/or progression of a disease in a subject.
  • the method comprises providing a series of biological samples over a time period from the subject; isolating disease-derived
  • microvesicles comprising RNAs from the series of biological samples; determining an amount of one or more of the RNAs in each of the biological samples from the series; and determining any measurable change in the amounts of the one or more RNAs in each of the biological samples from the series to thereby evaluate treatment efficacy and/or progression of the disease in the subject. Any changes in the amounts of measured RNAs over the time period can be used to predict clinical outcome, determine whether to initiate or continue the therapy for the disease, and whether a current therapy is effectively treating the disease. For example, a first time point can be selected prior to initiation of a treatment and a second time point can be selected at some time after initiation of the treatment.
  • RNA levels can be measured in each of the samples taken from different time points and qualitative and/or quantitative differences noted. A change in the amounts of one or more of the measured RNA levels from the first and second samples can be correlated with prognosis, theranosis, determining treatment efficacy, and/or progression of the disease in the subject.
  • correlated and “correlating,” as used herein in reference to the use of diagnostic and prognostic RNA levels associated with a disease refer to comparing the presence or quantity of the RNA levels in a subject to its presence or quantity in subjects known to suffer from a disease, or in subjects known to be free of the disease, i.e. "normal subjects” or “control subjects.” For example, a level of one or more RNAs in a biological sample can be compared to a RNA level for each of the specific RNAs tested and determined to be correlated with a disease.
  • RNA level(s) is said to have been correlated with a diagnosis; that is, the skilled artisan can use the RNA level(s) to determine whether the subject suffers from the disease and respond accordingly.
  • the sample's RNA level(s) can be compared to control RNA level(s) known to be associated with a good outcome (e.g., the absence of disease), such as an average level found in a population of normal subj ects .
  • a diagnostic or prognostic RNA level is correlated to a disease by merely its presence or absence.
  • a threshold level of a diagnostic or prognostic RNA level can be established, and the level of the RNA in a subject sample can simply be compared to the threshold level.
  • RNA level(s) can be determined at an initial time, and again at a second time.
  • an increase in the RNA level(s) from the initial time to the second time can be diagnostic of the disease, or a given prognosis.
  • a decrease in the RNA level(s) from the initial time to the second time can be indicative of the disease, or a given prognosis.
  • the degree of change of one or more RNA level(s) can be related to the severity of the disease and/or timeline of disease progression and future adverse events.
  • comparative measurements can be made of the same RNA level(s) at multiple time points, one can also measure given RNA level(s) at one time point, and second RNA level(s) at a second time point, and a comparison of these levels can provide diagnostic information.
  • determining the prognosis refers to methods by which the skilled artisan can predict the course or outcome of a condition in a subject.
  • the term “prognosis” can refer to the ability to predict the course or outcome of a condition with up to 100% accuracy, or predict that a given course or outcome is more or less likely to occur based on the presence, absence or levels of a biomarker.
  • the term “prognosis” can also refer to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a subject exhibiting a given condition, when compared to those individuals not exhibiting the condition.
  • the chance of a given outcome may be very low (e.g., ⁇ 1%), or even absent.
  • the chance of a given outcome may be higher.
  • a prognosis is about a 5% chance of a given expected outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, or about a 95% chance.
  • RNA level(s) e.g., quantity of one or more RNAs in a sample
  • a control level can signal that a subject is more likely to suffer from a disease than subjects with a level less than or equal to the control level, as determined by a level of statistical significance.
  • a change in RNA level(s) from baseline levels can be reflective of subject prognosis, and the degree of change in marker level can be related to the severity of adverse events.
  • Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, incorporated herein by reference in its entirety. Exemplary confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. When performing multiple statistical tests, e.g., determining differential expression of a panel of RNA levels, p values can be corrected for multiple comparisons using techniques known in the art.
  • a threshold degree of change in the level of a prognostic or diagnostic RNA level(s) can be established, and the degree of change in the level of the indicator in a biological sample can simply be compared to the threshold degree of change in the level.
  • a preferred threshold change in the level for RNA level(s) of the presently disclosed subject matter is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 60%, about 75%, about 100%, or about 150%.
  • a "nomogram" can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages.
  • the identity and relative quantity of RNAs in a sample can be used to provide RNA profiles for a particular sample.
  • An RNA profile for a sample can include information about the identities of RNAs contained in the sample, quantitative levels of RNAs contained in the sample, and/or changes in quantitative levels of RNAs relative to another sample.
  • an RNA profile for a sample can include information about the identities, quantitative levels, and/or changes in quantitative levels of RNAs associated with a particular disease.
  • a preferred subject is a vertebrate subject.
  • a preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal.
  • a mammal is most preferably a human.
  • the term "subject" includes both human and animal subjects.
  • veterinary therapeutic uses are provided in accordance with the presently disclosed subject matter.
  • domesticated fowl i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans.
  • livestock including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.
  • the presently disclosed subject matter provides for the determination of the amount of disease-derived micro vesicle RNAs correlated with disease within biological fluids of a subject, and in particular, from serological samples from a subject, such as for example blood. This provides the advantage of biological samples for testing that are easily acquired from the subject.
  • the amount of one or more RNAs of interest in the biologic sample can then be determined using any of a number of methodologies generally known in the art and compared to RNA control levels.
  • the "amount" of one or more RNAs determined refers to a qualitative (e.g., present or not in the measured sample) and/or quantitative (e.g., how much is present) measurement of the one or more RNAs.
  • the "control level” is an amount (including the qualitative presence or absence) or range of amounts of one or more RNAs found in a comparable biological sample in subjects not suffering from disease. As one non-limiting example of calculating the control level, the amount of one or more RNAs of interest present in a normal biological sample (e.g., blood) can be calculated and extrapolated for whole subjects.
  • RNA levels from microvesicles in a biological sample is microarray technique, which is a powerful tool applied in gene expression studies.
  • the technique provides many polynucleotides with known sequence information as probes to find and hybridize with the complementary strands in a sample to thereby capture the complementary strands by selective binding.
  • probe binding refers to a measure of the capacity of a probe to hybridize to a target polynucleotide with specificity.
  • the probe comprises a polynucleotide sequence that is complementary, or essentially complementary, to at least a portion of the target polynucleotide sequence.
  • complementary are those which are base-pairing according to the standard Watson-Crick complementarity rules.
  • complementary sequences means nucleic acid sequences which are substantially complementary, as can be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein.
  • a particular example of a contemplated complementary nucleic acid segment is an antisense oligonucleotide.
  • the probe can be 100% complementary with the target polynucleotide sequence.
  • the probe need not necessarily be completely complementary to the target polynucleotide along the entire length of the target polynucleotide so long as the probe can bind the target polynucleotide with specificity and capture it from the sample.
  • Stringent temperature conditions will generally include temperatures in excess of 30° C, typically in excess of 37° C, and preferably in excess of 45° C.
  • Stringent salt conditions will ordinarily be less than 1,000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. Determining appropriate hybridization conditions to identify and/or isolate sequences containing high levels of homology is well known in the art. For the purposes of specifying conditions of high stringency, preferred conditions are a salt concentration of about 200 mM and a temperature of about 45° C.
  • microarray can profile hundreds and thousands of polynucleotides simultaneously with high throughput performance.
  • Microarray profiling analysis of mRNA expression has successfully provided valuable data for gene expression studies in basic research. And the technique has been further put into practice in the pharmaceutical industry and in clinical diagnosis. With increasing amounts of RNA data becoming available, and with accumulating evidence of the importance of RNA in gene regulation, microarray becomes a useful technique for high through-put RNA studies.
  • RNA correlated with disease can be carried out separately or simultaneously with multiple polynucleotide probes within one test sample. For example, several probes can be combined into one test for efficient processing of a multiple of samples and for potentially providing greater diagnostic and/or prognostic accuracy.
  • probes can be combined into one test for efficient processing of a multiple of samples and for potentially providing greater diagnostic and/or prognostic accuracy.
  • one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same subject.
  • Such testing of serial samples can allow the identification of changes in RNA levels over time. Increases or decreases in RNA levels, as well as the absence of change in levels, can provide useful information about the disease status.
  • a panel consisting of polynucleotide probes that selectively bind disease-derived microvesicle RNAs correlated with one or more diseases can be constructed to provide relevant information related to the diagnosis or prognosis of disease and management of subjects with disease.
  • a panel can be constructed, for example, using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, or 1,000 individual polynucleotide probes.
  • a panel comprises more than 1,000 individual polynucleotide probes.
  • the analysis of a single probe or subsets of probes comprising a larger panel of probes could be carried out by one skilled in the art to optimize clinical sensitivity or specificity in various clinical settings. These include, but are not limited to ambulatory, urgent care, critical care, intensive care, monitoring unit, in-subject, out- subject, physician office, medical clinic, and health screening settings. Furthermore, one skilled in the art can use a single probe or a subset of additional probes comprising a larger panel of probes in combination with an adjustment of the diagnostic threshold in each of the aforementioned settings to optimize clinical sensitivity and specificity.
  • the clinical sensitivity of an assay is defined as the percentage of those with the disease that the assay correctly predicts, and the specificity of an assay is defined as the percentage of those without the disease that the assay correctly predicts.
  • determining the amount of the one or more RNAs comprises labeling the one or more RNAs.
  • the labeled RNAs can then be captured with one or more polynucleotide probes that each selectively bind the one or more RNAs.
  • label and “labeled” refer to the attachment of a moiety, capable of detection by spectroscopic, radiologic, or other methods, to a probe molecule.
  • label or “labeled” refer to incorporation or attachment, optionally covalently or non-covalently, of a detectable marker into/onto a molecule, such as a polynucleotide.
  • Various methods of labeling polypeptides are known in the art and can be used.
  • labels for polynucleotides include, but are not limited to, the following: radioisotopes, fluorescent labels, heavy atoms, enzymatic labels or reporter genes, chemiluminescent groups, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for antibodies, metal binding domains, epitope tags, etc.).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • RNA levels using polynucleotide probes can be carried out in a variety of physical formats as well.
  • the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples.
  • single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion.
  • the plurality of polynucleotide probes are each bound to a substrate.
  • the substrate comprises a plurality of addresses. Each address can be associated with at least one of the polynucleotide probes of the array.
  • An array is "addressable" when it has multiple regions of different moieties (e.g., different polynucleotide sequences) such that a region (i.e., a "feature” or “spot” of the array) at a particular predetermined location (i.e., an "address") on the array will detect a particular target or class of targets (although a feature may incidentally detect non-targets of that feature).
  • Array features are typically, but need not be, separated by intervening spaces.
  • the "target” RNA can be referenced as a moiety in a mobile phase (typically fluid), to be detected by probes (“target probes”) which are bound to the substrate at the various regions.
  • Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods.
  • Methods of depositing obtained biopolymers include, but are not limited to, loading then touching a pin or capillary to a surface, such as described in U.S. Pat. No. 5,807,522 or deposition by firing from a pulse jet such as an inkjet head, such as described in PCT publications WO 95/251 16 and WO 98/41531, and elsewhere.
  • the in situ fabrication methods include those described in U.S. Pat. No.
  • the array regions will often be exposed to one or more reagents to form a suitable layer on the surface that binds to both the substrate and biopolymer or biomonomer.
  • the array regions will also typically be exposed to the oxidizing, deblocking, and optional capping reagents.
  • Determining the amount of disease-derived microvesicle RNAs can alternatively, or in addition to microarray analysis, comprise using real-time polymerase chain reaction (PCR), for example such as is disclosed in detail in the present Examples.
  • PCR real-time polymerase chain reaction
  • RT-PCR Real-time PCR
  • the methods of the invention comprise providing a biological sample from a subject and isolating microvesicles comprising RNAs from the biological sample.
  • the biological sample can be a bodily fluid such as described herein, e.g., plasma or serum.
  • An amount of one or more of the RNAs is then determined and compared to one or more RNA control levels.
  • the subject can then be diagnosed with having or being at risk of disease if there is a measurable difference in the amount of the one or more RNAs from the microvesicles as compared to the one or more RNA control levels.
  • the levels of the one or more RNAs can also be used to provide a prognosis or a theranosis, such as to classify the subject as a likely responder or non-responder to a treatment or to monitor the efficacy of a treatment over time.
  • methods can include predicting response to a treatment in a subject, or predicting non-response of a treatment in a subject.
  • the control levels can be the levels of the one or more RNAs in a control sample that does not have or is not at risk of having disease, e.g., the control sample can be from a healthy subject.
  • a control can also be the level of the one or more RNAs at a different time point. For example, a decrease in the level of one or more RNA in a subject over time may indicate a response to a treatment.
  • the presently-disclosed subject matter is inclusive of uses of reagents as described herein and reagents known to those of ordinary skill in the art to carry out the methods as disclosed herein and described in the claims.
  • the presently-disclosed subject matter further includes kits that include reagents as described herein and reagents known to those of ordinary skill in the art to carry out the methods as disclosed herein and described in the claims.
  • kits which are useful for practicing embodiments of the methods as described herein.
  • a kit is provided, which is useful for determining a presence or an amount of one or more micro RNAs, which includes a probe for determining the presence or amount of each of one or more mircroRNAs in a sample.
  • the probe(s) are polynucleotides.
  • a primer pair is used to determine the amount of the one or more microRNAs.
  • the probe(s) is provided on a substrate.
  • the kit includes a probe for each of at least 2, 3, 4, 5, 6, 7, ,8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 R As.
  • kits of the presently-disclosed subject matter further include a reference standard sample to obtain a presence or amount of the one or more microRNAs for use as a control to which the sample (e.g., sample from the subject) can be compared.
  • the systems further include control data of a presence or level of the one or more microRNAs for use as a control to which the sample (e.g., sample from the subject) can be compared.
  • the systems further include reference data for one or more clinicopathologic features useful for characterizing a disease- of-interest.
  • the standard sample or the control data can be selected from: a standard sample or control data a disease; a standard sample or control data for ovarian cancer; a standard sample or control data for lung cancer; a standard sample or control data for adverse pregnancy outcome; a standard sample or control data for non- cancer; a standard sample or control data for a responder; and a standard sample or control data for a nonresponder.
  • microR As miRNAs or miRs
  • miRNAs or miRs are identified with reference to names assigned by the miRBase Registry (available at www.mirbase.org).
  • the sequences and other information regarding the identified miRNAs as set forth in the miRBase Registry are expressly incorporated by reference as are equivalent and related miRNAs present in the miRBase Registry or other public databases.
  • Also expressly incorporated herein by reference are all annotations present in the miRBase Registry associated with the miRNAs disclosed herein. Unless otherwise indicated or apparent, the references to the miRBase Registry are references to the most recent version of the database as of the filing date of this Application (i.e., mirBase 19, released August 1, 2012).
  • long non-coding RNAs disclosed herein are identified with reference to names assigned by the Long Non-Coding RNA Database (IncRNAdb) (available at www.lncrnadb.org).
  • the sequences and other information regarding the identified lncRNAs as set forth in the IncRNAdb are expressly incorporated by reference as are equivalent and related lncRNAs present in the IncRNAdb or other public databases.
  • Also expressly incorporated herein by reference are all annotations present in the IncRNAdb associated with the lncRNAs. Unless otherwise indicated or apparent, the references to the IncRNAdb are references to the most recent version of the database as of the filing date of this Application (i.e., IncRNAdb, released July 3, 2012).
  • the term "about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
  • ranges can be expressed as from “about” one particular value, and/or to "about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 1 1, 12, 13, and 14 are also disclosed.
  • Example 1 NUMERATION AND CHARACTERIZATION OF CIRCULATING VESICLES IN OVARIAN CANCER
  • NTA Nanoparticle Tracking analysis
  • Fractions 15, 16, and 17 contained the peak of the void volume.
  • NTA demonstrated a very narrow size range of vesicles (Figure 2B).
  • NTA submicron analysis
  • SPA submicron analysis
  • dynamic light scattering were compared with electron microscopic analysis of vesicles.
  • Phenotype of cell-derived vesicles A major limitation of DLS and SPA, as well as standard NTA, is that while they can objectively define the vesicle size range, they cannot define the "phenotype" of these vesicles.
  • NanoSight LM10 equipped with the 405- nm blue-violet laser and more sensitive camera to detect fluorescent particles, quantum dots attached to antibodies can be used identify specific subsets of vesicles. The instrument was initially calibrated using lOOnm and 200nm fluorescent beads, which can be easily discriminated. Antibodies reactive with either CD63 (exosomes marker) or EpCAM (marker of vesicles derived from epithelial tumors) were conjugated with quantum dots.
  • the labeled vesicle samples were analyzed on the NanoSight LM10, first in light scatter mode and then in fluorescence mode.
  • Panel A shows the NTA profile in light scatter mode and then fluorescence for CD63 (Panel B) and EpCAM (Panel C).
  • the size ranges of the CD63 -labeled vesicles are similar, with peaks in the region of lOOnm and 180nm whether measured in light scatter or fluorescence mode.
  • EpCAM on the various size ranges of vesicles indicates their tumor origin. Vesicles both larger and smaller than lOOnm exhibit CD63, the marker for exosomes, thus the published definition of exosomes as ranging only from 50-100nm may not be accurate.
  • the sample presented in Figure 6A was the NTA total of untreated vesicles, while Panel B presents the same sample treated with 0.5% Triton XI 00 for 5 minutes at room temperature and reanalyzed under the same conditions. Based on NTA, the total number of particles was diminished approximately 10-fold. Particle size analysis was further performed using Coulter Model N4 Plus particle size analyzer in PBS at room temperature (SDP analysis, 17 bins in the range from 1-lOOOnm at 90 degrees) using weight analysis. The sample in Figure 6C presents the SPA distribution of the same sample. The values observed ranged from 80- 120nm. Panel D was the same sample treated with 1% Tween 20 for 5 minutes at room temperature and reanalyzed in the same conditions. Based on SPA, the apparent weight average size of the particles shifted from 100 to ⁇ 10 nm (range 5-20 nm).
  • Example 2 ASSOCIATION AND SELECTIVITY OF MIRNA IN
  • miRNA was isolated from circulating tumor-derived exosomes using mirVana isolation kit. This total small RNA fraction was utilized for miRNA profiling as defined by qRT-PCR microarray analysis. Initial analyses were performed by cancer- specific arrays from SABiosciences. The small RNA -enriched fraction was extracted from the isolated exosomes. Using specific primers, presence and expression level of mature miRNAs was analyzed by TaqMan miRNA Assay (Applied Biosystems) under conditions defined by the supplier. LMW RNA was isolated from exosomes isolated from 1ml of sera using the mirVana miRNA Extraction Kit and quantified by the RiboGreen kit.
  • Single-stranded cDNA will be synthesized from 5.5ng of total RNA in 15 ⁇ 1 reaction volume by using the TaqMan MicroRNA Reverse Transcription Kit (AB). The reactions will be incubated first at 16°C for 30 min and then at 42°C for 30 min. The reactions will be inactivated by incubation at 85 °C for 5 min. Each cDNA generated will be amplified by quantitative PCR by using sequence-specific primers from the TaqMan microR A Assays Human Panel on a Agilent M3005P. The 20 ⁇ 1 PCR mix will include ⁇ of 2 Universal PCR Master Mix, 2 ⁇ 1 of each 10* TaqMan MicroRNA Assay Mix and 1.5 ⁇ 1 of reverse transcription (RT) product.
  • RT reverse transcription
  • the reactions will be incubated at 95 °C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
  • the threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold (0.2). All signals with CT ⁇ 37.9 will be manually set to undetermined.
  • the relative quantity (RQ) of the target miRNAs will be estimated by the ACT study by using as reference (exogenous control) for each preparation. Each sample will be run in duplicate and each PCR experiment will include two non-template control wells. From this analyses, exosomal miRNAs that were previously reported to be specifically up- regulated in ovarian cancer cells were examined ( Figure 7). Similarly, those exosomal miRNAs shown to be specifically down-regulated were examined.
  • One objective of this study was to define miRNA signatures that might differentiate early and late stage ovarian cancer.
  • serum specimens of patients with Stage I, II or III serous papillary adenocarcinoma of the ovary were evaluated.
  • the small RNA-enriched fraction was extracted from the isolated exosomes.
  • presence and expression level of mature miRNAs was analyzed by TaqMan miRNA Assay (Applied Biosystems) under conditions defined by the supplier.
  • LMW RNA was isolated from exosomes isolated from 1ml of sera using the mirVana miRNA Extraction Kit and quantified by the RiboGreen kit.
  • Single-stranded cDNA will be synthesized from 5.5ng of total RNA in 15 ⁇ 1 reaction volume by using the TaqMan MicroRNA Reverse Transcription Kit (AB). The reactions will be incubated first at 16°C for 30 min and then at 42°C for 30 min. The reactions will be inactivated by incubation at 85°C for 5 min. Each cDNA generated will be amplified by quantitative PCR by using sequence- specific primers from the TaqMan microRNA Assays Human Panel on a Agilent M3005P. The 20 ⁇ 1 PCR mix will include ⁇ of 2x Universal PCR Master Mix, 2 ⁇ 1 of each 10x TaqMan MicroRNA Assay Mix and 1.5 ⁇ 1 of reverse transcription (RT) product.
  • RT reverse transcription
  • the reactions will be incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
  • the threshold cycle is defined as the fractional cycle number at which the fluorescence passes the fixed threshold (0.2). All signals with CT ⁇ 37.9 will be manually set to undetermined.
  • Example 3 EXOSOMAL LONG NON-CODING RNA (LNCRNA) IN LUNG CANCER
  • This study analyzed IncRNA within ovarian tumor cell lines and in vitro released exosomes, as well as within exosomes derived from patients with serous papillary adenocarcinoma of the ovary.
  • ovarian tumor cells cells were grown in HyClone Serum- free media (SFM). After 3 days, media was removed and centrifuged at 400xg to remove cells and at 10,000xg to remove cell debris. The supernatant was concentrated 10-fold and microvesicles isolated by chromatography using Sepharose 2B. The void volume (vesicle fraction) was treated with Trizol to isolate total RNA.
  • SFM HyClone Serum- free media
  • RNA fraction was analyzed for specific IncRNAs using the LncRNA profiler qPCR array (Systems Biosciences). Similarly, the tumor cells were directly extracted with Trizol to isolate the RNA fraction and the IncRNAs were analyzed using the LncRNA profiler array.
  • exosomes were isolated from pooled normal human sera by ExoQuick and total RNA was isolated by Trizol and analyzed in parallel.
  • Exosomes isolated from the media of cultured tumor cells contain RNA populations.
  • One population identified here was IncRNA. Comparison of IncRNA profiles between tumor cells and their released exosomes reveal selectivity on the IncRNAs appearing in the exosomes. Of the 90 IncRNAs examined, 3 exhibited greater than 10-fold increase in the exosome population.
  • IncRNA are defined with specific regulatory activity, representative IncRNAs were compared between the cells and their released exosomes. For IncRNAs exhibiting epigenetic silencing, the CT value of ANRIL in cells was 23.80 compared to 26.44 in exosomes. Among IncRNAs exhibiting splicing regulation, the CT value of
  • MALAT-1 in cells was 31.84 compared to 32.85 in exosomes.
  • CT value of GAS-5 in cells was 31.72 compared to 29.86 in exosomes.
  • CT value of BACE1AS in cells was 34.14 compared to 34.86 in exosomes.
  • LncRNAs were detected within the exosomes isolated from the peripheral circulation of patients. While exosomal IncRNAs can be detected in both cancer patients and normal controls, the IncRNA profiles of cancer patients exhibit profiles distinct from normal ( Figure 11A). Of the 90 IncRNA analyzed, an increase of greater than 20-fold was observed in 58 IncRNAs in cancer patient-derived exosomes (versus control). In contrast, a decrease of 10-fold or greater was observed in 20 IncRNAs in cancer patients versus controls. Among IncRNAs exhibiting epigenetic silencing, HOTAIR exhibited a 42.85-fold increase in cancer- derived exosomes versus controls.
  • MALAT-1 was increased 24.7-fold in tumor-derived exosomes compared with controls.
  • GAS-5 was elevated 30.4-fold in cancer patient-derived exosomes versus controls.
  • BACE1AS was elevated 10,262-fold in patient exosomes versus controls.
  • LncRNAs contribute to genetic regulatory roles, including imprinting, epigenetic regulation, cell cycle control, nuclear and cytoplasmic trafficking, transcription, splicing, cell differentiation and apoptosis. Within tumors, the misexpression of IncRNAs contributes to cancer development and progression.
  • IncRNAs such as MALAT-1 (metastasis-associated in lung adenocarcinoma transcript) were identified in ovarian cancer and demonstrated to be critical in early stage development. Here the present inventors demonstrate the elevation of this IncRNA in exosomes from ovarian cancer patients. The stability of exosomes in the peripheral circulation and the unique profile of IncRNAs suggest their ideal utility as a diagnostic biomarker.
  • Example 4 EXOSOMAL LONG NON-CODING RNA (LNCRNA) IN LUNG CANCER
  • IncRNAs were compared between the cells and their released exosomes.
  • CT values of IncRNA within cells and the IncRNA within exosomes were the same (ANRIL cells 23.80 vs. 26.44 exosomes.
  • the IncRNA profiles of cancer patients were distinct from normal ( Figure 1 IB). An increase of greater than 20-fold was observed in 58 IncRNAs in cancer patient-derived exosomes.
  • IncRNAs distinct from non-cancer controls. Some IncRNAs, such as MALAT-1 (metastasis-associated in lung adenocarcinoma transcript) were identified in serum of NSCLC patients. The stability of exosomes in the peripheral circulation and the unique profile of IncRNAs suggest their ideal utility as a diagnostic biomarker.
  • Miscarriage occurs in an estimated 10 to 15 percent of all pregnancies less than 20 weeks gestation.
  • Recurrent miscarriage is classically defined as the occurrence of three or more consecutive losses of clinically recognized pregnancies prior to the 20 th week of gestation, exclusive of molar and ectopic pregnancies.
  • Prospective studies have assessed the risks of subsequent miscarriage after one loss to be 15 percent, rising to 17 to 31 percent after two losses, and 25 to 46 percent after three or more losses.
  • Most providers will initiate an evaluation for recurrent pregnancy loss after two or more consecutive miscarriages.
  • the pathophysiology of RPL is complex with many unknown contributing factors and mechanisms.
  • Endometrial lymphocytes of recurrent spontaneous aborters express distinct immune - phenotypic profiles that antedate implantation and suggest that endometrial immunologic conditions are intrinsically altered in recurrent aborters.
  • Activation of T-lymphocytes during pregnancy can result in one of two different cytokine profiles: Th2 secreted cytokines (i.e. IL- 4, IL-5, and IL-10) that suppress cellular immunity and Thl secreted cytokines (i.e. IFN- ⁇ , IL-2, and TNF-a) that induce cellular immunity.
  • Th2 secreted cytokines i.e. IL- 4, IL-5, and IL-10
  • Thl secreted cytokines i.e. IFN- ⁇ , IL-2, and TNF-a
  • An increase in the ratio of Th2 cytokines to Thl cytokines is associated with successful pregnancy and that a decrease in this ratio is associated with recurrent pregnancy loss.
  • Clinical studies
  • miRNA was isolated from circulating exosomes using mirVana isolation kits. This total small RNA fraction was utilized for miRNA profiling as defined by qRT-PCR microarray analysis. Initial analyses were performed by cancer-specific arrays from SABiosciences. The small RNA-enriched fraction was extracted from the isolated exosomes. Using specific primers, presence and expression level of mature miRNAs was analyzed by TaqMan miRNA Assay (Applied Biosystems) under conditions defined by the supplier. LMW RNA was isolated from exosomes isolated from 1ml of sera using the mirVana miRNA Extraction Kit and quantified by the RiboGreen kit.
  • Single-stranded cDNA will be synthesized from 5.5ng of total RNA in 15 ⁇ 1 reaction volume by using the TaqMan MicroRNA Reverse Transcription Kit (AB). The reactions will be incubated first at 16°C for 30 min and then at 42°C for 30 min. The reactions will be inactivated by incubation at 85 °C for 5 min. Each cDNA generated will be amplified by quantitative PCR by using sequence-specific primers from the TaqMan microRNA Assays Human Panel on a Agilent M3005P. The 20 ⁇ 1 PCR mix will include ⁇ of 2 Universal PCR Master Mix, 2 ⁇ 1 of each 10x TaqMan MicroRNA Assay Mix and 1.5 ⁇ 1 of reverse transcription (RT) product.
  • RT reverse transcription
  • the reactions will be incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60°C for 1 min.
  • the threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold (0.2). All signals with CT >37.9 will be manually set to undetermined.
  • the relative quantity (RQ) of the target miRNAs will be estimated by the ACT study by using as reference (exogenous control) for each preparation. Each sample will be run in duplicate and each PCR experiment will include two non-template control wells ( Figure 12).
  • the heat map presents the levels of all microRNA tested. Based on this data, 15 microRNAs are elevated in patients exhibiting RPL, while 30 are decreased compared to normal term delivering pregnancies ( Figure 13).
  • Cell-derived vesicles and exosomes are potential markers of human disease. This could include the identification of elevated circulating exosomes and the presence of specific exosome "cargo.” However, their use in diagnostic tests requires an objective and high throughput method of defining their size, concentration, and phenotype in biological fluids.
  • NTA nanoparticle tracking analysis
  • the size distribution of these unfractionated vesicles from cancer patients ranged from approximately 50 to 300nm in diameter. Patients with benign disease and controls exhibited a similar size range; however, they possessed a greater percentage of vesicles within the 200-300nm range (versus cancer).
  • the "gold standard" for defining the size and characteristics of exosomes is electron microscopy to demonstrate the presence of cup-shaped vesicles, ranging from 50- lOOnm. This size range was initially defined using exosomes derived from normal lymphocytes. One issue is that size can be modified by sample preparation for EM and the size distribution can be skewed by the image area selected for evaluation (subjective). Since EM studies use pelleted vesicles, image fields where individual vesicles can be visualized may be a limiting factor in this selection. In general, light scattering methodologies report a larger size range than EM and evaluate objectively the total vesicle populations. This study compared these methodologies using chromatographic ally isolation vesicles.
  • DLS and SPA while providing an objective size distribution of the entire vesicle population, do not define the concentration of the vesicles.
  • a limitation of DLS and SPA is that while they can objectively define the vesicle size range, they cannot define the "phenotype" of these vesicles.
  • NanoSight LM10 equipped with the 405- nm blue-violet laser and more sensitive camera to detect fluorescent particles
  • Vesicles both larger and smaller than lOOnm exhibit CD63 demonstrating that the published definition of exosomes as ranging only from 50-100nm may not be accurate.
  • the chromatographically isolated vesicles were initially analyzed by SPA and NT A, followed by a re-analysis after a 5 minute treatment with a non-ionic detergent.
  • SPA on the Coulter Model N4 Plus particle size analyzer
  • the values for vesicle size was observed to range from 80-100nm.
  • Tween 20 following treatment with 1% Tween 20 for 5 minutes at room temperature
  • the reanalysis shown that the average size of the particles shifted ⁇ 10nm (range 5-20 nm).
  • the same sample treated with 0.5% Triton XI 00 for 5 minutes at room temperature exhibited a 10-fold reduction.
  • Vesicle analyses based on light scattering and Brownian motion analyses allow quantitation of mean vesicle size and size distribution.
  • NTA has the additional advantage of defining concentration.
  • the disadvantage of SPA and DLS is that they are unable to determine the phenotype of the vesicles. Since biological fluids and clinical specimens comprise mixtures of vesicles derived from many different cell types, it is essential to be able to determine the cell of origin and to understand their biological function, the molecules that they express on their surface.
  • the present inventors have now isolated exosomes from the peripheral circulation of patients with ovarian cancer or from non-tumor-bearing controls by size exclusion chromatography. Further isolation of tumor-specific exosomes by a lectin based method revealed a subpopulation of vesicles within the peripheral circulation with the mean diameter at 72nm. The number of vesicular particles was also determined by the Nanosight analysis, demonstrating the presence of 2.46 x 10 10 vesicles/ml. Analyses of the "cargo" of this enriched tumor derived exosome fraction will allow us to specifically define markers of tumor status and not markers of the patient's normal cell response to the tumor. This is critical as the host response is likely to be non-specific (such as a pro-inflammatory immune response) and could result in significant false positive results.
  • RNAs contribute to genetic regulatory roles, including imprinting, epigenetic regulation, cell cycle control, nuclear and cytoplasmic trafficking, transcription, splicing, cell differentiation and apoptosis.
  • LncRNAs contribute to genetic regulatory roles, including imprinting, epigenetic regulation, cell cycle control, nuclear and cytoplasmic trafficking, transcription, splicing, cell differentiation and apoptosis.
  • the misexpression of IncRNAs contributes to cancer development and progression. Cancer patients exhibit a significant increased level of circulating vesicles in the range of 50-200nm, which exhibited markers confirming their exosome origin. These exosomes contain IncRNA and their profiles were distinct from non-cancer controls.
  • IncRNAs such as MALAT-1 (metastasis-associated in lung adenocarcinoma transcript) were identified in ovarian cancer and demonstrated to be critical in early stage development. Here the elevation of this IncRNA in exosomes from ovarian cancer patients is demonstrated ( Figure 11). The stability of exosomes in the peripheral circulation and the unique profile of IncRNAs suggest their ideal utility as a diagnostic biomarker.
  • Petricoin EF Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM. Use of proteomic patterns in serum to identify ovarian cancer. Lancet, 359:572-577,
  • Activated platelets release two types of membrane vesicles: Microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha granules. Blood 94, 3791-3799.
  • Taylor DD Black PH (1986) Shedding of plasma membrane fragments: Neoplastic and developmental importance. In: Developmental Biology (vol. 3), Steinberg MS (ed) pp 33-57. Plenum Press: New York. Taylor DD, Black PH (1987) Neoplastic and developmental importance of
  • metalloproteinases as a potential screening test for gynecologic malignancies.
  • Taylor DD Sullivan SA, Eblen AC, Gercel-Taylor C.Modulation of T-cell CD3- zeta chain expression during normal pregnancy. J Reprod Immunol, 54: 15-31, 2002. Taylor DD, Gercel-Taylor C. Tumor-derived exosomes as mediates of T-cell signaling defects. Brit J Cancer, 92: 305-31 1, 2005
  • Ambros V The functions of animal microRNAs. Nature 431 :350-355, 2004.
  • RNA expression detected by oligonucleotide microarrays system establishment and expression profiling in human tissues. Genome Res 14: 2486-2494.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des procédés de caractérisation de maladies et d'évaluation du traitement et/ou de la progression de maladies sur la base de la détermination de quantités d'un ou plusieurs ARN issus de microvésicules dans un échantillon biologique.
PCT/US2014/025675 2013-03-13 2014-03-13 Arn associé à un exosome en tant que marqueur de diagnostic Ceased WO2014160032A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361780397P 2013-03-13 2013-03-13
US61/780,397 2013-03-13

Publications (1)

Publication Number Publication Date
WO2014160032A1 true WO2014160032A1 (fr) 2014-10-02

Family

ID=51625314

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/025675 Ceased WO2014160032A1 (fr) 2013-03-13 2014-03-13 Arn associé à un exosome en tant que marqueur de diagnostic

Country Status (1)

Country Link
WO (1) WO2014160032A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104357443A (zh) * 2014-11-25 2015-02-18 中国科学院生物物理研究所 一种用于膀胱癌筛查的长链非编码rna的检测及其应用
CN104388543A (zh) * 2014-10-27 2015-03-04 中南大学 长链非编码rna loc401317的原位杂交探针、试剂及其应用
CN104388542A (zh) * 2014-10-27 2015-03-04 中南大学 长链非编码rna loc401317原位杂交探针的应用方法
CN105200156A (zh) * 2015-10-30 2015-12-30 中南大学 长链非编码rna基因loc553103的应用
CN105200152A (zh) * 2015-10-30 2015-12-30 中南大学 长链非编码rna基因loc553103在制备鼻咽癌预后制剂上的应用
WO2016089732A3 (fr) * 2014-12-01 2016-08-04 University Of South Florida Méthodes et compositions utilisables en vue du diagnostic et de la prise en charge du diabète et du syndrome métabolique
CN106702009A (zh) * 2017-03-06 2017-05-24 北京泱深生物信息技术有限公司 一种与肺腺癌相关的基因的应用
WO2017121988A1 (fr) * 2016-01-12 2017-07-20 The University Of York Production de protéines recombinantes
CN107447008A (zh) * 2017-08-24 2017-12-08 南京医科大学 用于诊断不明原因复发性流产的增强子rna组合、引物组及应用和试剂盒
CN111235274A (zh) * 2020-01-18 2020-06-05 山西医科大学第一医院 喉鳞癌血清外泌体标志物筛选方法和外泌体来源miR-941应用
WO2020142436A1 (fr) * 2019-01-02 2020-07-09 Arizona Board Of Regents On Behalf Of The University Of Arizona Systèmes et procédés de caractérisation et de traitement d'une maladie
WO2021013592A1 (fr) * 2019-07-19 2021-01-28 Fundación Para La Investigación Biomédica Del Hospital Universitario La Paz (Fibhulp) Procédé de détermination de la réponse au traitement d'un patient atteint d'un carcinome pulmonaire non à petites cellules (nsclc)
CN112813167A (zh) * 2021-02-08 2021-05-18 江苏省肿瘤医院 标志物linc01977及其应用
CN114277141A (zh) * 2020-03-30 2022-04-05 中国医学科学院肿瘤医院 用于肺癌诊断的试剂盒、装置及方法
CN118291606A (zh) * 2023-09-15 2024-07-05 山东大学齐鲁医院 Rna组合及其在复发性流产诊断标记物中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009015357A1 (fr) * 2007-07-25 2009-01-29 University Of Louisville Research Foundation, Inc. Micro arn associé à l'exosome en tant que marqueur de diagnostic
WO2009021322A1 (fr) * 2007-08-16 2009-02-19 The Royal Institution For The Advancement Of Learning/Mcgill University Microvésicules issues d'une cellule tumorale
WO2009155505A2 (fr) * 2008-06-20 2009-12-23 Proxy Life Science Holdings, Inc. Compositions et procédés à base de microvésicules
US20100196426A1 (en) * 2008-02-01 2010-08-05 The General Hospital Corporation Use of microvesicles in diagnosis and prognosis of medical diseases and conditions
WO2012031008A2 (fr) * 2010-08-31 2012-03-08 The General Hospital Corporation Matières biologiques liées au cancer dans des microvésicules

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009015357A1 (fr) * 2007-07-25 2009-01-29 University Of Louisville Research Foundation, Inc. Micro arn associé à l'exosome en tant que marqueur de diagnostic
WO2009021322A1 (fr) * 2007-08-16 2009-02-19 The Royal Institution For The Advancement Of Learning/Mcgill University Microvésicules issues d'une cellule tumorale
US20100196426A1 (en) * 2008-02-01 2010-08-05 The General Hospital Corporation Use of microvesicles in diagnosis and prognosis of medical diseases and conditions
WO2009155505A2 (fr) * 2008-06-20 2009-12-23 Proxy Life Science Holdings, Inc. Compositions et procédés à base de microvésicules
WO2012031008A2 (fr) * 2010-08-31 2012-03-08 The General Hospital Corporation Matières biologiques liées au cancer dans des microvésicules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, QUAN ET AL.: "Long non-coding RNAs (IncRNAs) and viral infections.", BIOMEDICINE & PHARMACOTHERAPY, vol. 3, no. 1, 1 March 2013 (2013-03-01), pages 34 - 42 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104388543A (zh) * 2014-10-27 2015-03-04 中南大学 长链非编码rna loc401317的原位杂交探针、试剂及其应用
CN104388542A (zh) * 2014-10-27 2015-03-04 中南大学 长链非编码rna loc401317原位杂交探针的应用方法
CN104357443B (zh) * 2014-11-25 2017-03-15 中国科学院生物物理研究所 一种用于膀胱癌筛查的长链非编码rna的检测及其应用
CN104357443A (zh) * 2014-11-25 2015-02-18 中国科学院生物物理研究所 一种用于膀胱癌筛查的长链非编码rna的检测及其应用
US10724097B2 (en) 2014-12-01 2020-07-28 University Of South Florida Methods and compositions for diagnosis and management of diabetes and metabolic syndrome
WO2016089732A3 (fr) * 2014-12-01 2016-08-04 University Of South Florida Méthodes et compositions utilisables en vue du diagnostic et de la prise en charge du diabète et du syndrome métabolique
CN105200156B (zh) * 2015-10-30 2017-12-22 中南大学 长链非编码rna基因loc553103的应用
CN105200156A (zh) * 2015-10-30 2015-12-30 中南大学 长链非编码rna基因loc553103的应用
CN105200152A (zh) * 2015-10-30 2015-12-30 中南大学 长链非编码rna基因loc553103在制备鼻咽癌预后制剂上的应用
CN105200152B (zh) * 2015-10-30 2018-02-16 中南大学 长链非编码rna基因loc553103在制备鼻咽癌预后制剂上的应用
WO2017121988A1 (fr) * 2016-01-12 2017-07-20 The University Of York Production de protéines recombinantes
US11268101B2 (en) 2016-01-12 2022-03-08 The University Of York Recombinant protein production
CN106702009A (zh) * 2017-03-06 2017-05-24 北京泱深生物信息技术有限公司 一种与肺腺癌相关的基因的应用
CN107447008A (zh) * 2017-08-24 2017-12-08 南京医科大学 用于诊断不明原因复发性流产的增强子rna组合、引物组及应用和试剂盒
WO2020142436A1 (fr) * 2019-01-02 2020-07-09 Arizona Board Of Regents On Behalf Of The University Of Arizona Systèmes et procédés de caractérisation et de traitement d'une maladie
WO2021013592A1 (fr) * 2019-07-19 2021-01-28 Fundación Para La Investigación Biomédica Del Hospital Universitario La Paz (Fibhulp) Procédé de détermination de la réponse au traitement d'un patient atteint d'un carcinome pulmonaire non à petites cellules (nsclc)
CN111235274A (zh) * 2020-01-18 2020-06-05 山西医科大学第一医院 喉鳞癌血清外泌体标志物筛选方法和外泌体来源miR-941应用
CN114277141A (zh) * 2020-03-30 2022-04-05 中国医学科学院肿瘤医院 用于肺癌诊断的试剂盒、装置及方法
CN114277141B (zh) * 2020-03-30 2022-09-02 中国医学科学院肿瘤医院 外泌体cda、mboat2等在肺癌诊断中的应用
CN112813167A (zh) * 2021-02-08 2021-05-18 江苏省肿瘤医院 标志物linc01977及其应用
CN118291606A (zh) * 2023-09-15 2024-07-05 山东大学齐鲁医院 Rna组合及其在复发性流产诊断标记物中的应用

Similar Documents

Publication Publication Date Title
WO2014160032A1 (fr) Arn associé à un exosome en tant que marqueur de diagnostic
JP5156829B2 (ja) 診断マーカーとしてのエキソソーム関連マイクロｒｎａ
US20150018227A1 (en) Microrna biomarkers
Jovčevska Genetic secrets of long-term glioblastoma survivors
Eluard et al. The alternative RelB NF-κB subunit is a novel critical player in diffuse large B-cell lymphoma
JP5624470B2 (ja) ヒト末梢血微小胞におけるｍｉＲＮＡの発現およびその使用
US20200063208A1 (en) Exosomal biomarkers for diagnosis and prognosis of cancer and related methods
CN111630183B (zh) 透明细胞肾细胞癌生物标志物
Husna et al. Identification of melanoma‐specific exosomal miRNAs as the potential biomarker for canine oral melanoma
US20200308655A1 (en) Plasma Microribonucleic Acids as Biomarkers for Endometriosis and Endometriosis-Associated Ovarian Cancer
WO2013063439A1 (fr) Caractérisation d'un mélanome
Karatas et al. The role of miR-145 in stem cell characteristics of human laryngeal squamous cell carcinoma Hep-2 cells
WO2020157070A1 (fr) Nouveaux biomarqueurs et profils de diagnostic pour le cancer de la prostate
Luo et al. The role of Exosomes in the Pathogenesis of Nasopharyngeal Carcinoma and the involved Clinical Application
Oladejo et al. MicroRNAome: potential and veritable immunomolecular therapeutic and diagnostic baseline for lingering bovine endometritis
Li et al. USP15 facilitates the progression of bladder cancer by amplifying the activation of the NF-κB signaling pathway
CN114480656B (zh) Slc12a3和/或slc12a9在作为葡萄膜黑色素瘤治疗和预后检测指标中的应用
EP4615995A1 (fr) Méthodes impliquant la détection du gène 6 stimulé par tnf (tsg-6) pour améliorer les réponses anti-tumorales à une immunothérapie chez des patients atteints de cancer
US20150080229A1 (en) Plasma microribonucleic acids as biomarkers for endometriosis and endometriosis-associated ovarian cancer
WO2020198633A1 (fr) Exosomes d'origine nucléaire et procédés d'utilisation de ceux-ci
Namazi et al. Down-regulation of miR-193b-3p and miR-376a-3p in Chronic Lymphocytic Leukemia
Gong et al. hsa-miR-124-3p: A New Potential Prognostic Target For Multiple Myeloma
Chamandi miRNA profiling in breast cancer: putative novel biomarkers for diagnosis, prognosis, or therapeutics
Wu et al. The Tumor‐to‐Endothelial Transfer of FTO Promotes Vascular Remodeling and Metastasis in Nasopharyngeal Carcinoma
Alix-Panabieres et al. Liquid Biopsy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14775183

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14775183

Country of ref document: EP

Kind code of ref document: A1